Methods and compositions for screening for altered cellular phenotypes

ABSTRACT

The invention relates to methods and compositions useful for screening for altered cellular phenotypes using an inducible expression system to enrich for and detect the altered phenotypes and, more particularly, relates to screening libraries of candidate bioactive agents, for example, nucleic acids and peptides, in cells using an regulatable expression system to enrich for a subpopulation of cells having an altered phenotype due to the presence of a candidate bioactive agent.

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/076,624, filed May 12, 1998 (pending).

FIELD OF THE INVENTION

[0002] The invention relates to methods and compositions useful forscreening for altered cellular phenotypes using an inducible expressionsystem to enrich for and detect the altered phenotypes. In particular,the invention relates to screening libraries of candidate bioactiveagents, for example, nucleic acids and peptides, in cells using anregulatable expression system to enrich for a subpopulation of cellshaving an altered phenotype due to the presence of a candidate bioactiveagent.

BACKGROUND OF THE INVENTION

[0003] Inducible expression systems have been developed to facilitatethe analysis of gene function in cells and to facilitate the developmentof effective treatments using gene therapy. These expression systemsattempt to control nucleic acid expression by using inducible eukaryoticpromoters that are responsive to inducers such as hormones (Lee et al.(1981) Nature 294:228-232; Hynes et al. (1981) Proc. Natl. Acad. Sci.USA 78:2038-2042; Klock et al. (1987) Nature 329:734-736; Israel &Kaufman (1989) Nucl. Acids Res. 17:2589-2604); heavy metal ions (Mayo etal. (1982) Cell 29:99-108; Brinster et al. (1982) Nature 296:3942;Searle et al. (1985) Mol. Cell. Biol. 5:1480-1489); or heat shock (Noueret al. (1991) in Heat Shock Response, e.d. Nouer, L., CRC, Boca Raton,Fla., pp 167-220). However, these expression systems are problematicbecause the eukaryotic promoters can exhibit a high level of basalexpression in the non-induced state; the inducers can promotepleiotropic effects; and the level of induction can be low.

[0004] In order to overcome these problems, inducible eukaryoticexpression systems utilizing prokaryotic regulatory elements have beendeveloped. The rationale for using prokaryotic regulatory elements in aeukaryotic expression system is based on the theory that effectorsmodulating the activity of such prokaryotic regulatory elements wouldnot be responsive to eukaryotic cellular components. Therefore,pleiotropic effects would be eliminated. An example of such a system isthe lac operator regulatable expression system. In this system,expression of sequences operably linked to the lac operator isconstitutively induced (or “turned on”) by a LacR-VP16 fusion proteinand is repressed (or “turned off”) in the presence ofisopropyl--D-thiogalactopyranoside (IPTG) (Labow et al. (1990), citedsupra). In another lac inducible system, the binding of LacR-VP16 to theoperator sequence is enhanced by increasing the temperature of thecells. However, IPTG in eukaryotic cells is an inefficient inducer ofnucleic acid expression and must be used at concentrations nearcytotoxic levels. Furthermore, increasing the temperature of the cellsis likely to promote pleiotropic effects in the cells. Thus, there is aneed for a more efficient inducible regulatory system that exhibitsrapid and high level induction of nucleic acid expression; is highlyresponsive to a specific exogenous inducer; and exhibits low levels ofexpression in the uninduced state.

[0005] The teteracycline (Tet) inducible system utilizes entirelyprokaryotic components and, thus, pleiotropic effects are avoided(Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen etal. (1995) Science 268:1766-1769). In this system, the inducer is anintegral component of a transactivator that binds to the induciblepromoter and drives expression of a nucleic acid of interest. Thus, theintermediate steps in the induction pathway are largely eliminated andthe control of expression is highly specific and tightly controlled bycontact with the inducer. The Tet-controlled expression system thereforeprovides an effective means for turning off and turning on nucleic acidexpression in cells and, thereby, allowing for the regulated expressionof a nucleic acid in cells.

[0006] With the advent of functional genomics, large numbers of nucleicacids encoding genes of unknown function have been isolated and cloned.Consequently, there is a critical need to develop rapid and highlyefficient methods for screening large numbers of candidate nucleic acidsfor analysis of gene function and to identify potential targets fordevelopment of therapeutic agents.

[0007] One approach for studying gene function is to regulate theexpression of a nucleic acid in cells and look for a correspondingalteration in cellular phenotype. Consequently, rapid and highlyefficient methods of screening diverse populations of cells for analtered phenotype due to the inducible expression of a candidate nucleicacid is highly desirable and useful for the analysis of nucleic acidfunction and target discovery.

[0008] Thus, it is an object of the present invention to provide rapidand highly efficient methods of screening populations of cells for analtered cellular phenotype due to the presence of a candidate bioactiveagent, using an inducible expression system that permits the regulatedexpression of candidate nucleic acids encoding a candidate bioactiveagent and permits controlled expression of the candidate nucleic acid ata defined level.

SUMMARY OF THE INVENTION

[0009] In accordance with the objects described above, the presentinvention provides methods and compositions for screening for an alteredcellular phenotype using an inducible expression system to enrich forand detect cells having an altered phenotype due to the presence of acandidate bioactive agent. In the methods of the present invention,detection of cells having an altered cellular phenotype is achieved byselection of cells responsive to the induction and repression ofexpression of a nucleic acid sequence encoding the candidate bioactiveagent. The cells having an altered cellular phenotype are enriched bymultiple rounds of selection.

[0010] The invention provides methods and compositions for screeningcandidate bioactive agents useful as targets for drug discovery, byaccessing molecules and targets within living cells and provides for thedirect selection of those bioactive agents with desired phenotypiceffects. Further, the methods and compositions of the present inventionare particularly useful for high throughput screening of candidatebioactive agents capable of altering a cellular phenotype.

[0011] The invention provides compositions for use in the methods of thepresent invention. Specifically, the invention provides populations ofcells having a parent phenotype and comprising a nucleic acid encoding afirst element expressed in the cells. The invention further provideslibraries of fusion nucleic acids, where the fusion nucleic acidscomprise a second element that is regulatable by the first element. Thefusion nucleic acids further comprise a nucleic acid sequence operablylinked to the second element. The nucleic acid sequence encodes acandidate bioactive agent. In addition, the invention provides a thirdelement that induces or represses the expression of the nucleic acidsequence encoding the candidate bioactive agent. Thus, the inventionprovides two approaches for screening for an altered cellular phenotype:in a first approach the third element induces expression; and in asecond approach the third element represses expression.

[0012] Using either approach, in the methods of the present invention,the cells having an altered phenotype due to the presence of thecandidate bioactive agent are distinguished and detected based on theirresponsiveness to the induction and repression (in either order) ofexpression of the nucleic acid sequence encoding the candidate bioactiveagent, by the third element. The responsive cells have the parentphenotype when expression of the nucleic acid sequence is repressed andhave an altered phenotype when expression of the nucleic acid sequenceis induced. The nonresponsive cells can be distinguished by having aphenotype that is not responsive to the induction and repression ofexpression of the nucleic acid sequence.

[0013] Using the first approach, the invention provides methods ofscreening for an altered cellular phenotype comprising the steps of: a)providing a population of cells having a parent phenotype and comprisinga nucleic acid encoding a first element inducibly or constitutivelyexpressed in the cells; b) introducing a library of fusion nucleic acidsinto the population of cells, where the fusion nucleic acids comprise asecond element that is regulatable by the first element and furthercomprise a nucleic acid sequence encoding a candidate bioactive agent;c) inducing the expression of the nucleic acid sequence by contactingthe cells with a third element; d) collecting a first subpopulation ofcells having an altered phenotype; e) repressing the expression of thenucleic acid by modulating the contacting of the third element with thefirst subpopulation of cells; f) collecting a second population of cellshaving a parent phenotype; g) inducing the expression of the nucleicacid sequence by contacting the third element with the secondsubpopulation of cells; and g) detecting a third subpopulation of cells.

[0014] Using the second approach, the invention provides methods ofscreening for an altered cellular phenotype comprising the steps of: a)providing a population of cells having a parent phenotype and comprisinga nucleic acid encoding a first element inducibly or constitutivelyexpressed in the cells; b) introducing a library of fusion nucleic acidsinto the population of cells, where the fusion nucleic acids comprise asecond element that is regulatable by the first element and furthercomprise a nucleic acid sequence encoding a candidate bioactive agent;c) inducing the expression of the nucleic acid sequence by expressingthe first element; d) collecting a first subpopulation of cells havingan altered phenotype; e) repressing the expression of the nucleic acidby contacting a third element with the first subpopulation of cells; f)collecting a second population of cells having a parent phenotype; g)inducing the expression of the nucleic acid sequence modulating thecontacting the third element with the second subpopulation of cells; andh) detecting a third subpopulation of cells.

[0015] Using either approach, the invention provides methods ofscreening for an altered cellular phenotype further comprisingcollecting the responsive cells. The methods also further compriserepeating the induction or repression of expression and detecting theresponsive cells and, further, collecting the cells to enrich for asubpopulation of cells having the altered phenotype. The inducing orrepressing of expression, followed by detecting and, further, bycollecting a subpopulation of cells responsive to the induction andrepresssion can be repeated multiple times to obtain a desired level ofenrichment and detection of a subpopulation of cells having an alteredcellular phenotype due to the presence of a candidate bioactive agent.

[0016] Examples of a first element include, but are not limited to, atransactivator. In one aspect, the first element comprises atetracycline-dependent transactivator (tTA) or a reversetetracycline-dependent transactivator (rtTA). The first element can beinducible; expressed constitutively, expressed stably or transiently;and expressed in trans or in cis relative to the nucleic acid sequence.In one aspect, the fusion nucleic acid further comprises the nucleicacid encoding the first element. Examples of a second element include,but are not limited to, an operator sequence. In one aspect, theoperator sequence comprises a tetracycline operator sequence (TetO). Inanother aspect the second element is an oligomer of a TetO sequence.Examples of a third element include, but are not limited to, a moleculethat induces or represses expression of the nucleic acid sequence. Inone aspect, the molecule comprises tetracycline or analogues thereof,e.g., doxycycline (Dox).

[0017] In one aspect, the first element comprises a reversetetracycline-dependent activator (rtTA) and expression of the nucleicacid sequence is induced by contacting the third element with the cells,and is repressed by modulating the contacting of the third element withthe cells.

[0018] In another aspect, the first element comprises atetracycline-dependent activator (tTA) and expression of the nucleicacid sequence is repressed by contacting the third element with thecells, and is induced by modulating the contacting of the third elementwith the cells.

[0019] Examples of candidate bioactive agents include, but are notlimited to, nucleic acids and polypeptides. Further examples ofbioactive agents are cyclic peptides, RNA, antisense RNA, and DNA.Additional examples of nucleic acid sequences encoding a candidatebioactive agent, include but are not limited to, random nucleic acidsequences, and biased random nucleic acid sequences. Examples of nucleicacid sequences encoding a candidate bioactive agent, also include butare not limited to, full-length cDNA sequences, subsequences of afull-length cDNA, and antisense sequences of a full-length cDNA. Anotherexample of a nucleic acid sequence encoding a candidate bioactive agentis a nucleic acid sequence encoding an amino acid sequence that isin-frame or out-of-frame as compared to the open reading frame (ORF)encoded by the amino acid sequence of a full-length cDNA.

[0020] In one aspect, the present invention provides methods comprisingthe steps of: a) providing a population of cells having a parentphenotype and comprising a nucleic acid encoding a first element that isexpressed in the cells, for example, a reverse tetracycline-dependenttransactivator (rtTA); b) introducing into the population of cells alibrary of fusion nucleic acids, where the nucleic acids each comprise asecond element that is regulatable by the first element, and a nucleicacid sequence that is operably linked to the first element, where thenucleic acid sequence encodes a candidate bioactive agent; c) inducingthe expression of the nucleic acid sequence by contacting a thirdelement with the population of cells, wherein the population of cells isexpressing the first element; d) collecting a first subpopulation ofcells having an altered phenotype; e) repressing the expression of thenucleic acid sequence by modulating the contacting of the third elementwith the first subpopulation of cells; f) collecting a secondsubpopulation of cells having the parent phenotype; g) inducing theexpression of the nucleic acid sequence by contacting the third elementwith the second subpopulation of cells; and h) detecting a thirdsubpopulation of cells having the altered phenotype.

[0021] In an additional aspect, the method further comprises: i)collecting the third subpopulation of cells having an altered phenotype;j) repressing the expression of the nucleic acid sequence by modulatingthe contacting of the third element with the third subpopulation ofcells; and k) detecting a fourth subpopulation of cells having theparent phenotype.

[0022] In another additional aspect, the method further comprises: l)collecting the fourth subpopulation of cells having the parentphenotype; m) inducing the expression of the nucleic acid sequence bycontacting the third element with the fourth subpopulation of cells; andn) detecting a fifth subpopulation of cells having the alteredphenotype.

[0023] In another aspect, the invention provides a method of screeningfor cells having an altered phenotype comprising the steps of: a)providing a population of cells having a parent phenotype and comprisinga nucleic acid encoding a first element, for example, atetracycline-dependent transactivator; b) introducing into thepopulation of cells a library of fusion nucleic acids, where the nucleicacids each comprise a second element that is regulatable by the firstelement, and a nucleic acid seq uence that is operably linked to thefirst element, where the nucleic acid sequence encodes a candidatebioactive agent; c) inducing the expression of the nucleic acid sequenceby expressing the first element in the population of cells; d)collecting a first subpopulation of cells having an altered phenotype;e) repressing the expression of the nucleic acid by contacting a thirdelement with the first subpopulation of cells; f) collecting a secondsubpopulation of cells having the parent phenotype; g) inducing theexpression of the nucleic acid sequence by modulating the contacting ofthe third element with the second subpopulationof cells; and h)detecting a third subpopulation of cells having the altered phenotype.

[0024] In an additional aspect, the method further comprises: i)collecting the third subpopulation of cells having the alteredphenotype; j) repressing the expression of the nucleic acid sequence bycontacting the third element with the third subpopulation of cells; andk) detecting a fourth subpopulation of cells having the parentphenotype.

[0025] In a further additional aspect, the method further comprises: l)collecting the fourth subpopulation of cells having the parentphenotype; m) inducing the expression of the nucleic acid sequence bymodulating the contacting of the third element with the fourthsubpopulation of cells; and n) detecting a fifth subpopulation of cellshaving said altered phenotype.

[0026] In one aspect, the first element comprises a reversetetracycline-dependent activator (rtTA); the second element comprises anoligomer of a tetracycline operator sequence (TetO); and the thirdelement comprises tetracycline or doxycycline.

[0027] In another aspect, the first element comprises atetracycline-dependent activator (rtTA); said second element comprisesan oligomer of a tetracycline operator sequence (TetO); and said thirdelement comprises tetracycline (Tet) or doxycycline (Dox).

[0028] In a another aspect, the library of fusion nucleic acidscomprises about 10³ to 10⁹ different said nucleic acid sequences, or 10⁴to 10³ different random nucleic acid sequences. In a further aspect, thefusion nucleic acids are each a component of a retroviral vector.

[0029] In another aspect, the fusion nucleic acids further comprise asequence encoding a reporter protein, and this sequence is operablylinked to the nucleic acid sequence encoding the candidate bioactiveagent. Examples of a reporter protein include, but are not limited to,an autofluorescent protein, for example, a green fluorescent protein(GFP) from Aqueorea, or a Renilla species.

[0030] In a further aspect, the cells are collected byfluorescence-activated cell sorting (FACS)

[0031] In a further aspect, the parent phenotype of the cells is due tothe presence of a stimulator and the cells comprise a stimulator. In apreferred embodiment, the stimulator induces the parent phenotype.Examples of stimulators include, but are not limited to, cytokines,ligands or antibodies against cells surface receptors, growth factors,hormones, peptides, neuropeptides,drugs or compounds, LPS, viruses,bacteria, and so on. In a preferred embodiment, the stimulator isinterleukin 4 (IL-4), a cytokine, that has various biologicalactivities, for example, IL-4 induces germlne epsilon promoter(transcription) in B cells. In another preferred embodiment, thestimulator is anti-TCR (T cell receptor), a stimulator that activates Tcell activation as monitored by CD69 upregulation.

[0032] In another aspect, the cells having an altered phenotype aremammalian cells. Examples of mammalian cells having an alteredphenotype, include but are not limited to, rodent cells and human cells.

[0033] In another aspect, the altered phenotype is the modulation of a Tcell surface marker, for example, CD3, CD25, CD28, CD40L, CD69, CD95, orCD95L. Further examples of an altered phenotype, include but are notlimited to, the modulation of cell cycle regulation, exocytosis, IgEsecretion, IgE switching, antigen-induced B cell differentiation,antigen-induced B cell isotyping, apoptosis, angiogenesis, and T cellreceptor (TCR) activation.

BRIEF DESCRIPTION OF THE FIGURES

[0034]FIG. 1 schematically depicts the cell line BH1-4 and HBEGFDiphtheria toxin selection in the cell line.

[0035]FIG. 2 schematically depicts the cell line BH2-A5 and HBEGFDiphtheria toxin selection in the cell line.

[0036]FIG. 3 depicts the conventional screening method.

[0037]FIG. 4 schematically depicts five different retroviral vectorswhich were constructed as positive controls for the screening assay, andare the encoded GFP, SOCS1, STAT6Δ, and/or ires are operably linked to apromoter in the retroviral vector: the retroviral vector cGFP containsthe GFP; the retroviral vector SOCS1-ires-GFP contains, from 5′ to 3′(and operably linked), SOCS1, internal ribosomal entry site (IRES), andGFP; the retroviral vector GFP-SOCS1 contains, from 5′ to 3′ (andoperably linked), GFP-SOCS1 fusion; the retroviral vectorSTAT6Δ-ires-GFP contains, from 5′ to 3′ (and operably linked), STAT6Δ,ires, and GFP; and the retroviral vector GFP-STAT6Δ contains, from 5′ to3′ (and operably linked), GFP-STAT6Δ fusion.

[0038]FIG. 5 depicts the assay of the five retroviral vectors describedin FIG. 4, in BH1-4 cells.

[0039]FIG. 6 depicts the effects of SOCS1 and STAT6 on IL4/diphtheria(IL4/dip) induced death of BH1-4 cells 6 days post infection for each ofthe five retroviral vectors described in FIG. 4.

[0040]FIG. 7 depicts the same results as depicted in FIG. 6 except thatthe vertical axis indicates the number of cells, and the horizontal axisindicates the amount of GFP fluorescence.

[0041]FIG. 8 depicts a screening assaying using spiked cell.

[0042]FIG. 9 schematically depicts the IL4-dip selection assay usingBH1-4 cells and depicts the results of the assay starting with a 1:10dilution of the SOCS1-infected cells.

[0043]FIG. 10 depicts the results for screening assays using BH1-4 cellsstarting with the 1:10, 1:100, 1:1,000, and 1:10,000 dilution of theSOCS1-infected cells.

[0044]FIG. 11 depicts the results of screening assays for BH1-4 andBH2-A5 cells starting with the 1:10, 1:100, 1:1,000, and 1:10,000dilution of the spiked cells.

[0045]FIG. 12 depicts the results of selection beginning with naïvecells in a first round of selection with IL4-Dip and then subjecting thesurviving cells from the first round of selection to a second round ofselection with IL4-Dip.

[0046]FIG. 13 depicts a screening assay using a novel cell line BH2-A5Twith the peptide library BFP-C20 encoded by retroviral vectors.

[0047]FIG. 14 depicts the cell line BH2-A5T (or “A5T” or “A5T-4”) whichexpresses a Tet regulated transactivator (tTA or Tet transactivator) andallows for the Tet regulated expression of candidate bioactive agentsintroduced into the cells.

[0048]FIG. 15 depicts histograms indicating the amount of GFPfluorescence, in an experiment where IL4 responders were selected in thepresence and absence of Dox in BH2-A5T cells spiked with cells infectedwith the retroviral construct TRA-SOCS1-ires-GFP.

[0049]FIG. 16 depicts the round to round enrichment for cells expressingthe known inhibitor SOCS1 by induction and repression ofTRA-SOCS1-ires-GFP expression in BH2-A5T-4 cells using dox and sortingof the altered and parental cellular phenotype.

[0050]FIG. 17 depicts histograms indicating the amount of GFPfluorescence indicative of TRA-SOCS1-ires-GFP expression in cells afterthe first round of selection.

[0051]FIG. 18 depicts histograms showing the “Sort for IL4 responders”step in FIG. 16.

[0052]FIG. 19 depicts histograms showing the “Turn inhibitor expressionback on” step in FIG. 16.

[0053]FIG. 20 depicts for the 1:10,000 dilution of cells, an 6.1%enrichment resulting from the first round of selection; for the secondround of IL4/dip selection, a 88% enrichment from the de-doxed Left Gatecells and a 97% enrichment from de-doxed Right Gate cells from FIG. 19;and for the 1:100,000 dilution of cells, an 0.3% enrichment resultingfrom the first round of selection; for the second round of IL4/dipselection, a 26% enrichment from the de-doxed Left Gate cells and 47%enrichment from de-doxed Right Gate cells resulting from the sorting ofthe cells cultured in the absence of Dox (FIG. 19).

[0054]FIG. 21 schematically depicts an example of a timeline (in days)for a screening assay of the present invention, where the assay involvesa first round of selection and sorting; a second round of selection andsorting; and thereafter single cell clones are grown. The single cellclones are then subjected to selection and FACS assays, the nucleic acidencoding the bioactive agent (e.g., a peptide inhibitor of IL4signaling/induced expression) is then rescued and the phenotype isreconfirmed, e.g., by infecting naive cells with the rescued nucleicacid and selection.

[0055]FIG. 22 schematically depicts an example of a timeline (in days)for a screening assay of the present invention (and as described forFIG. 21), where the complexity of the library of candidate bioactiveagents (e.g., a peptide library), and the fold enrichment for thealtered cellular phenotype, are indicated. Further, FIG. 22schematically depicts the histogram profile of GFP fluorescence of falsepositives due to hereditable background or stochastic non-hereditablebackground; as compared to the histogram profile of GFP fluorescence ofcells cultured in the presence (+Tet) or absence (−Tet) of Tet, after afirst round of selection.

[0056]FIG. 23 schematically depicts an example of a timeline (in days)for a screening assay of the present invention (and as described forFIG. 21), where the complexity of the library of candidate bioactiveagents (e.g., a peptide library), and the fold enrichment for thealtered cellular phenotype, are indicated. Further, after a second ofselection, cells are single cell cloned, aliquoted into microtiterplates, replica plated in duplicate microtiter plates and cultured inthe presence or absence of Dox, and the single clones are contacted withIL4 for three days and their GFP fluorescence measured by FACS.

[0057]FIG. 24 depicts the histogram profile of GFP fluorescence ofclones from a functional screen representing a BFP-peptide inhibitorclone, CR2 (left panel); a hereditable background clone (middle panel),and stochastic background clone (right panel), where the histograms fromthe clones cultured in the presence of Dox (+Dox) and the absence of Dox(−Dox) are overlayed.

[0058]FIG. 25 depicts the summary of the results from the peptidescreening in BH2-A5T-4 cells.

[0059]FIG. 26 depicts cell line and assay development. FIG. 26A depictsJurkat cells stimulated with anti-T cell receptor (TCR) antibody C305 at300 ng/ml and 24 hrs later, cells stained with anti-CD69APC and analyzedon a FACSCalibur. The dashed line indicates CD69 level beforestimulation and the solid line after stimulation. FIG. 26B depictsJurkat clone (4D9) with optimal CD69 expression profile infected with aretroviral construct which constitutively expresses a tetracyclinetransactivator protein (tTA) and a reporter construct which expressesLyt2 driven by a tetracycline responsive element (TRE). The tTA-Jurkatcell clone 4D9#32 was obtained by sorting for high Lyt2 expression inthe absence of Doxycycline (Dox) and low expression of Lyt2 in thepresence Doxycycline (10 ng/ml). The solid line indicates Lyt2 levelwith Dox and dashed line without Dox.

[0060]FIG. 27 depicts dominant negative mutants of ZAP-70 inhibitedTCR-induced CD69 expression. FIG. 27A depicts ZAP70 K1 and ZAP70 SH2(N+C) subcloned downstream of the internal ribosome entry site (IRES),followed by GFP in the Tet-regulated retroviral vector (TRE). FIG. 27Bdepicts after infecting tTA-Jurkat cells with retroviral constructscontaining IRES-GFP, ZAP70 KI-IRES-GFP, or ZAP70 SH2 (N+C)-IRES-GFP,cells left unstimulated or stimulated with anti-TCR antibody for 24hours. CD69 expression data were analyzed after gating on the GFP⁺population (infected population, shown in R1). The dashed line and thethin line indicate cells infected with IRES-GFP (vector) before andafter TCR stimulation, respectively, and the thick line indicates cellsinfected with ZAP70 KI-IRES-GFP (top panel) or ZAP70 SH2 (N+C)-IRES-GFP(bottom panel), both after TCR stimulation. FIG. 27C depicts afterinfecting tTA-Jurkat cells with retroviral constructs containingIRES-GFP (vector) or ZAP70 SH2 (N+C)-IRES-GFP, cells treated with Doxfor 6 days, and then left unstimulated or stimulated with anti-TCRantibody for 24 hrs. Addition of Dox turned off GFP expression, as shownby the loss of GFP⁺ cells in the region R1. CD69 expression data wereanalyzed on the entire cell population. The dashed line and the thinline indicate cells infected with IRES-GFP (vector) before and after TCRstimulation, respectively, and the thick line indicates cells infectedwith ZAP70 SH2 (N+C)-IRE S-GFP after TCR stimulation. FIG. 27D depictstTA Jurkat cells containing different retroviral constructs (shown abovethe lanes) cultured in the absence (−) or presence (+) of Dox and lysed.Whole cell lysates were loaded (100:g each lane) and analyzed by Westernblotting using anti-ZAP70 antibody (Upstate Biotechnology catalog#05-253).

[0061]FIG. 28 depicts a screen for inhibitors of TCR-activation inducedCD69 expression. FIG. 28A depicts a scheme of the functional geneticscreen for inhibitors of TCR-activation induced CD69 expression. 3.5×10⁸cells were infected with pTRA-cDNA libraries. CD69^(low)CD3⁺ cellsrepresent cells expressing the lowest level of CD69 (bottom 3%) andstill retaining CD3 expression after TCR stimulation, whereasCD69^(high) cells are those expressing a high level of CD69 (top 10%)after grown with Dox and after TCR stimulation. Single cell cloning tookplace after at least 4 consecutive sorting of CD69^(low)CD3 ⁺ with orwithout the placement of sorting of CD69^(high) cells in between. FIG.28B depicts phenotypic enrichment via iterative cell sorting. 7.1×10⁸cells were sorted with high-speed flow sorters (MoFlo) after stimulationand staining with anti-CD69-APC and anti-CD3-PE. The sort gate was setat the equivalent of 1% of the control cells that were stimulated butwere never flow-sorted (shown as R2) to enrich for the CD69^(low)CD3⁺phenotype. After sorting, the desired cells were allowed to rest for aweek before another round of stimulation and sorting. With reiterativesorting, not only the desired population was enriched (R2 cells from 1%to 23.2%), but also the overall population demonstrated a reduced CD69level (shown as Y geo mean from >300 to 65). FIG. 28B depicts Doxregulation of the sorted population. Cells were split to two populationsafter the third round of sorting for the CD69^(low)CD3⁺ phenotype (shownas R2). One half of the cells were grown in the absence of Dox (top leftdot plot) while the other half in the presence of Dox (top right dotplot). A week later, CD69 expression was compared following anti-TCRstimulation. The dashed line indicates CD69 level without Dox and thesolid line with Dox.

[0062]FIG. 29 depicts the identification of clones with desired alteredphenotype. FIG. 29A depicts Individual clones grown in the presence orabsence of Dox for a week and then stimulated overnight with anti-TCRantibody. Cells were stained with anti-CD69-APC and analyzed on aFACSCalibur. The filled peaks indicate CD69 expression level in theabsence of Dox, when the cDNA hits were expressed. The open peaksindicate CD69 expression level in the presence of Dox, when the cDNAhits were not expressed. The geometric means of CD69 histograms are asfollows: 490.68 (+Dox) and 28.60 (−Dox) for clone 15; 658.45 (+Dox) and52.98 (−Dox) for clone 24; 553.46 (+Dox) and 40.09 (−Dox) for clone 64;433.44 (+Dox) and 82.2 (−Dox) for clone 116; 1235.77 (+Dox) and 17.68(−Dox) for clone 157; and finally, 245.81 (+Dox) and 26.43 (−Dox) forclone 194. The difference between filled and open peaks in any givenclone is represented by the Dox ratio of the CD69 geometric means (usingthe +Dox values divided by the −Dox values), which reflects thedependence of the altered phenotype on the cDNA expression. FIG. 29Bdepicts 2,828 cell clones were assayed for CD69 expression afterstimulation in the presence or absence of Dox. The geometric mean ofCD69 fluorescent units in the presence of Dox was divided by those inthe absence of Dox to give rise to the Dox Ratio for individual clones.A total of 1323 clones showed a Dox ratio of >1.5. The numbers of clonesshowing a Dox ratio between 1.5-10 was plotted against the Dox ratiothemselves to illustrate the population distribution. FIG. 29C depictsDNA oligonucleotide primers specific to the library vector were designed(BstXTRA5G and BstXTRA3D) and used in RT-PCR reactions. The RT-PCRproducts were analyzed in an agarose gel followed by ethidium bluestaining. Data from representative clones were shown along side the 1 kbDNA molecular weight ladder from New England BioLabs (Catalog #N3232S).

[0063]FIG. 30 depicts the functional transfer of the phenotype of knownTCR signaling molecules. Diagrams of proteins predicted from the cDNAinserts and those from the corresponding wild-type genes were shownabove the histograms. The left panel of histograms shows theDox-regulatable phenotype of the original cell clones. The original cellclones were grown in the presence or absence of Dox for a week and thenstimulated overnight with anti-TCR antibody. Cells were stained withanti-CD69-APC and analyzed by FACS. The filled peaks indicate CD69expression level in the absence of Dox, when the cDNA hits wereexpressed. The open peaks indicate CD69 expression level in the presenceof Dox, when the cDNA hits were not expressed. The Dox ratio was shownfor each original mutant clone. The right top and bottom panels ofhistograms show the phenotypes after expressing the cDNA inserts(followed by IRES-GFP) in a naive tTAJurkat population. After retroviralinfection, the tTA-Jurkat cells were either stimulated with the anti-TCRantibody (+″−TCR, solid line) or left unstimulated (−″−TCR, dashedline), and analyzed by FACS for CD69 induction after staining withanti-CD69-APC. The top right histogram in each group analyzed GFP⁻cells, which did not express the cDNA hit, whereas the bottom righthistogram in each group analyzed GFP⁺ cells, which expressed the cDNAhit. The following cDNA hits were analyzed: LCK (A), ZAP70 hit #1 (B),SYK (C), and PLCyl (D).

[0064]FIG. 31 depicts the functional transfer of phenotype of unknownTCR signaling molecules. Diagrams of proteins predicted from the cDNAinserts and those from the corresponding wild-type genes were shownabove the histograms. The left panel of histograms shows theDox-regulatable phenotypes of the original cell clones. The originalcell clones were grown in the presence or absence of Dox for a week andthen stimulated overnight with anti-TCR antibody. Cells were stainedwith anti-CD69-APC and analyzed by FACS. The filled peaks indicate CD69expression level in the absence of Dox, when the cDNA hits wereexpressed. The open peaks indicate CD69 expression level in the presenceof Dox, when the cDNA hits were not expressed. The Dox ratio was shownfor each original mutant clone. The right top and bottom panels ofhistograms show the phenotypes after expressing the cDNA inserts(followed by IRES-GFP) in a naive tTAJurkat population. After retroviralinfection, the tTA-Jurkat cells were either stimulated with the anti-TCRantibody (+″−TCR, solid line) or left unstimulated (−″−TCR, dashedline), and analyzed by FACS for CD69 induction after staining withanti-CD69-APC. The top right histogram in each group analyzed GFP⁻cells, which did not express the cDNA hit, whereas the bottom righthistogram in each group analyzed GFP⁺ cells, which expressed the cDNAhit. The following cDNA hits were analyzed: TCPTP (A), IL1 ORA (B),Integrin ″2 (C), and GG2-l (D).

[0065]FIG. 32 depicts the cDNA hits from the screening methods of thepresent invention inhibited T cell activation in human primary Tlymphocytes. FIG. 32A depicts retroviral infection of primary Tlymphocytes. Primary T lymphocytes were cultured on anti-CD3 andanti-CD28 coated wells for 3 days and then infected with the retroviralCRU5-GFP vector, where GFP was expressed from the constitutively activeretroviral LTR promoter. Cells were stained with anti-CD3-APC, or withanti-CD4-PE and anti-CD8-APC antibodies and analyzed by FACS. Thepercentage of cells in each quadrant is shown. FSC: forward scatter; andSSC: side scatter. FIG. 32B depicts human primary T lymphocytes wereinfected with vector alone (CRU5-GFP and CRU5-IRES-GFP or CIG) or withvector expressing the Lck and PLC(1 dominant negative (DN) hits. Theinfection rate was monitored by the percentage of GFP⁺ cells in M1. Thegeometric mean of GFP was shown above the marker. FIG. 32C depicts IL-2production was inhibited by LCK DN and PLC(1 DN proteins in primary Tlymphocytes. Infected primary T cells were allowed to rest and thensorted to give rise to GFP⁻ (filled bars) and GFP⁺ (open bars)populations. Equal numbers of cells were cultured in 96-well dish coatedwith anti-CD3 or anti-CD3+anti-C1)28 antibodies, cultured withoutantibodies or cultured with PMA+ionomycin. 40 hrs later the culturesupernatants were harvest and assayed for IL-2 production by ELISA usingcommercial reagents (R&D Systems).

[0066]FIG. 33 depicts an overview of identified molecular targets asdescribed in Example 7 and designated Table 2.

DETAILED DESCRIPTION OF THE INVENTION

[0067] The present invention provides methods and compositions forscreening for an altered cellular phenotype due to the presence of acandidate bioactive agent. The methods of the present invention providea significant improvement over conventional screening techniques becausethe methods allow the rapid and highly efficient screening of largenumbers of candidate bioactive agents in cells without the need tocollect or synthesize the candidate bioactive agents. In particular, themethods of the present invention afford significant advantages overconventional screening techniques because the methods permit the tightlycontrolled and highly specific expression of a candidate nucleic acid incells, and rapid detection of a subpopulation of cells highly enrichedfor a cellular phenotype corresponding to the controlled expression ofthe candidate nucleic acid. In particular, multiple rounds of selectioncan be performed to achieve the desired enrichment of cells having analtered phenotype corresponding to the expression of a candidate nucleicacid. Thus, the advantages of the methods of the present inventioninclude the ability to rapidly and efficiently screen diversepopulations of cells for an altered cellular phenotype corresponding tothe inducible expression of a candidate nucleic acid.

[0068] The invention provides compositions for use in the methods of thepresent invention. Specifically, the invention provides populations ofcells having a parent phenotype and comprising a nucleic acid encoding afirst element expressed in the cells. The first element is preferably atransactivator, and more preferably a Tet-dependent transactivator. Theinvention further provides libraries of fusion nucleic acids, where thefusion nucleic acids comprise a second element that is regulatable bythe first element. The second element preferably comprises an operatorsequence, and more preferably at least one Tet operator (TetO) sequence.The fusion nucleic acids further comprise a nucleic acid sequenceoperably linked to the second element. The nucleic acid sequencepreferably encodes a candidate bioactive agent. The candidate bioactiveagent is preferably a peptide or a nucleic acid. In addition, theinvention provides a third element that induces or represses theexpression of the nucleic acid sequence encoding the candidate bioactiveagent. The third element is preferably Tet or an analogue thereof. Thus,the invention provides two approaches for screening for an alteredcellular phenotype; in a first approach the third element induces(“turns on” expression; and in a second approach the third elementrepresses (“turns off”) expression.

[0069] Thus, generally, the invention works as follows. Cells exhibitingthe parent phenotype are induced to express the candidate agents, andthe cells are screened for those exhibiting an altered phenotype. Onceidentified and isolated as a first subpopulation, the expression of thecandidate agent is turned off, and the first subpopulation is againscreened for cells exhibiting the parent phenotype. This ensures ahigher level of confidence that the altered phenotype is due to thecandidate agent. This second subpopulation is again induced (orun-repressed, as the case may be) to express the candidate agent toresult in the altered phenotype. Thus, by screening for “off-on-off”(or, in some cases as outlined herein, “on-off-on”, etc., a morereproducible data set with a higher level of confidence is achieved. Inaddition, further reiterative rounds allow additional results. Ingeneral, this allows the elimination of non-responsive cells and thus“false positives”.

[0070] Using either approach, in the methods of the present invention,the cells having an altered phenotype due to the presence of thecandidate bioactive agent are distinguished and detected based on theirresponsiveness to the induction and repression of expression of thenucleic acid sequence encoding the candidate bioactive agent, by thethird element. The responsive cells have the parent phenotype whenexpression of the nucleic acid sequence is repressed and have an alteredphenotype when expression of the nucleic acid sequence is induced. Thenonresponsive cells can be distinguished by having a phenotype that isnot responsive to the induction and repression of expression of thenucleic acid sequence. As used herein, the term “parent phenotype”refers to the cellular phenotype of a cell in the uninduced state, i.e.,the expression of a nucleic acid sequence operably linked to a secondelement is turned off or repressed in the cell. As used herein, the term“altered phenotype” refers to the cellular phenotype of a cell in theinduced state, i.e., the expression of a nucleic acid sequence operablylinked to a second element is turned on or induced in the cell.

[0071] In a preferred embodiment, the parent phenotype is due to thepresence of a stimulator. In another preferred embodiment, thestimulator induces the parent phenotype. Examples of stimulatorsinclude, but are not limited to, cytokines, ligands or antibodiesagainst cells surface receptors, growth factors, hormones, peptides,neuropeptides, drugs or compounds, LPS, viruses, bacteria, and so on. Ina preferred embodiment, the stimulator is interleukin 4 (IL4), acytokine, that has various biological activities, for example, IL-4induces germlne epsilon promoter (transcription) in B cells. In anotherpreferred embodiment, the stimulator is anti-TCR (T cell receptor), astimulator that activates T cell activation as monitored by CD69upregulation.

[0072] In a preferred embodiment, the fusion nucleic acids furthercomprise a sequence encoding a reporter protein, and this sequence isoperably linked to the nucleic acid sequence encoding the candidatebioactive agent. Thus, the expression of the reporter proteincorresponds to the expression of the nucleic acid sequence encoding acandidate bioactive agent. Suitable reporter proteins are outlinedbelow.

[0073] Cells having an altered phenotype may be sorted from cells havinga parent phenotype using standard techniques known in the art for cellsorting. In the methods of the present invention, any chemical,physical, or physiological markers distinguishing an altered phenotypefrom a parent phenotype may be used to sort a population orsubpopulation of cells having a parent phenotype from a subpopulation ofcells having an altered phenotype. Thereby, a subpopulation of cellshaving an altered phenotype can be detected and collected. In apreferred embodiment, the fusion nucleic acids further comprise asequence encoding a reporter protein that is an autofluorescent protein,for example GFP from a Renilla species, and the cells having an alteredphenotype are sorted from the cells having a parent phenotype byfluorescence-activated cell sorting (FACS). In this embodiment, thesequence encoding the reporter protein is operably linked to a nucleicacid sequence encoding a candiate bioactive agent.

[0074] Using the first approach, the invention provides methods ofscreening for an altered cellular phenotype comprising the steps of: a)providing a population of cells having a parent phenotype and comprisinga nucleic acid encoding a first element inducibly or constitutivelyexpressed in the cells; b) introducing a library of fusion nucleic acidsinto the population of cells, where the fusion nucleic acids comprise asecond element that is regulatable by the first element and furthercomprise a nucleic acid sequence encoding a candidate bioactive agent;c) inducing the expression of the nucleic acid sequence by contactingthe cells with a third element; d) collecting a first subpopulation ofcells having an altered phenotype; e) repressing the expression of thenucleic acid by modulating the contacting of the third element with thefirst subpopulation of cells; f) collecting a second population of cellshaving a parent phenotype; g) inducing the expression of the nucleicacid sequence by contacting the third element with the secondsubpopulation of cells; and g) detecting a third subpopulation of cells.Using the second approach, the invention provides methods of screeningfor an altered cellular phenotype comprising the steps of: a) providinga population of cells having a parent phenotype and comprising a nucleicacid encoding a first element inducibly or constitutively expressed inthe cells; b) introducing a library of fusion nucleic acids into thepopulation of cells, where the fusion nucleic acids comprise a secondelement that is regulatable by the first element and further comprise anucleic acid sequence encoding a candidate bioactive agent; c) inducingthe expression of the nucleic acid sequence by expressing the firstelement; d) collecting a first subpopulation of cells having an alteredphenotype; e) repressing the expression of the nucleic acid bycontacting a third element with the first subpopulation of cells; f)collecting a second population of cells having a parent phenotype; g)inducing the expression of the nucleic acid sequence modulating thecontacting the third element with the second subpopulation of cells; andh) detecting a third subpopulation of cells.

[0075] Using either approach, the invention provides methods ofscreening for an altered cellular phenotype further comprisingcollecting the responsive cells. The methods also further compriserepeating the induction or repression of expression and detecting theresponsive cells and, further, collecting the cells to enrich for asubpopulation of cells having the altered phenotype. The inducing orrepressing of expression, followed by detecting and, further, bycollecting a subpopulation of cells responsive to the induction andrepresssion can be repeated multiple times to obtain a desired level ofenrichment and detection of a subpopulation of cells having an alteredcellular phenotype due to the presence of a candidate bioactive agent.

[0076] In a preferred embodiment, using the first approach, the presentinvention provides methods comprising the steps of: a) providing apopulation of cells having a parent phenotype and comprising a nucleicacid encoding a first element that is expressed in the cells, forexample, a reverse tetracycline-dependent transactivator (rtTA); b)introducing into the population of cells a library of fusion nucleicacids, where the nucleic acids each comprise a second element that isregulatable by the first element, and a nucleic acid sequence that isoperably linked to the first element, where the nucleic acid sequenceencodes a candidate bioactive agent; c) inducing the expression of thenucleic acid sequence by contacting a third element with the populationof cells, wherein the population of cells is expressing the firstelement; d) collecting a first subpopulation of cells having an alteredphenotype; e) repressing the expression of the nucleic acid sequence bymodulating the contacting of the third element with the firstsubpopulation of cells; f) collecting a second subpopulation of cellshaving the parent phenotype; g) inducing the expression of the nucleicacid sequence by contacting the third element with the secondsubpopulation of cells; and h) detecting a third subpopulation of cellshaving the altered phenotype.

[0077] In another preferred embodiment, using the first approach, themethod further comprises: i) collecting the third subpopulation of cellshaving an altered phenotype; j) repressing the expression of the nucleicacid sequence by modulating the contacting of the third element with thethird subpopulation of cells; and k) detecting a fourth subpopulation ofcells having the parent phenotype.

[0078] In another additional preferred embodiment, using the firstapproach, the method further comprises: l) collecting the fourthsubpopulation of cells having the parent phenotype; m) inducing theexpression of the nucleic acid sequence by contacting the third elementwith the fourth subpopulation of cells; and n) detecting a fifthsubpopulation of cells having the altered phenotype.

[0079] In another preferred embodiment, using the second approach, theinvention provides a method of screening for cells having an alteredphenotype comprising the steps of: a) providing a population of cellshaving a parent phenotype and comprising a nucleic acid encoding a firstelement, for example, a tetracycline-dependent transactivator; b)introducing into the population of cells a library of fusion nucleicacids, where the nucleic acids each comprise a second element that isregulatable by the first element, and a nucleic acid sequence that isoperably linked to the first element, where the nucleic acid sequenceencodes a candidate bioactive agent; c) inducing the expression of thenucleic acid sequence by expressing the first element in the populationof cells; d) collecting a first subpopulation of cells having an alteredphenotype; e) repressing the expression of the nucleic acid bycontacting a third element with the first subpopulation of cells; f)collecting a second subpopulation of cells having the parent phenotype;g) inducing the expression of the nucleic acid sequence by modulatingthe contacting of the third element with the second subpopulation ofcells; and h) detecting a third subpopulation of cells having thealtered phenotype.

[0080] In another embodiment, using the second approach, the methodfurther comprises: i) collecting the third subpopulation of cells havingthe altered phenotype; j) repressing the expression of the nucleic acidsequence by contacting the third element with the third subpopulation ofcells; and k) detecting a fourth subpopulation of cells having theparent phenotype.

[0081] In another embodiment, using the second approach, the methodfurther comprises: l) collecting the fourth subpopulation of cellshaving the parent phenotype; m) inducing the expression of the nucleicacid sequence by modulating the contacting of the third element with thefourth subpopulation of cells; and n) detecting a fifth subpopulation ofcells having said altered phenotype.

[0082] Accordingly, the invention provides methods for screening cellshaving an altered phenotype as compared to a parent phenotype. As usedherein, the term “parent phenotype” refers to the cellular phenotype ofa cell in the uninduced state, i.e., the expression of a nucleic acidsequence operably linked to a second element is turned off or repressedin the cell. As used herein, the term “altered phenotype” refers to thecellular phenotype of a cell in the induced state, i.e., the expressionof a nucleic acid sequence operably linked to a second element is turnedon or induced in the cell.

[0083] In a preferred embodiment, the parent phenotype is due to thepresence of a stimulator. A stimulator as used herein is an agent thatcan cause phenotypic changes. In another preferred embodiment, thestimulator induces the parent phenotype. Examples of stimulatorsinclude, but are not limited to, cytokines, ligands or antibodiesagainst cells surface receptors, growth factors, hormones, peptides,neuropeptides, drugs or compounds, LPS, viruses, bacteria, and so on(see e.g., Lorens et al. (2001) Pharmaceutical Biotechnology, pp.613-621). In a preferred embodiment, the stimulator is interleukin 4(IL-4), a cytokine, that has various biological activities, for example,IL-4 induces germlne epsilon promoter (transcription) in B cells. Inanother preferred embodiment, the stimulator is anti-TCR (T cellreceptor), a stimulator that activates T cell activation as monitored byCD69 upregulation.

[0084] The methods provide populations of cells. As will be appreciatedby those in the art, the type of cells used in the present invention canvary widely. Basically, any mammalian cells may be used, with mouse,rat, primate and human cells being particularly preferred, although aswill be appreciated by those in the art, modifications of the system bypseudotyping allows all eukaryotic cells to be used, preferably highereukaryotes. As is more fully described below, a screen will be set upsuch that the cells exhibit a selectable phenotype in the presence of abioactive agent. As is more fully described below, cell types implicatedin a wide variety of disease conditions are particularly useful becausethe methods of the present invention can be used to enrich for anddetect cells that exhibit an altered phenotype as a consequence of thepresence of a bioactive agent within the cell.

[0085] Accordingly, suitable cell types include, but are not limited to,tumor cells of all types (particularly melanoma, myeloid leukemia,carcinomas of the lung, breast, ovaries, colon, kidney, prostate,pancreas and testes), cardiomyocytes, endothelial cells, epithelialcells, lymphocytes (T-cell and B cell), mast cells, eosinophils,vascular intimal cells, hepatocytes, leukocytes including mononuclearleukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney,liver and myocyte stem cells (for use in screening for differentiationand de-differentiation factors), osteoclasts, chondrocytes and otherconnective tissue cells, keratinocytes, melanocytes, liver cells, kidneycells, and adipocytes. Suitable cells also include known research cells,including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos,etc. See the ATCC cell line catalog, hereby expressly incorporated byreference.

[0086] In one embodiment, the cells may be genetically engineered, thatis, contain exogeneous nucleic acid, for example, to contain targetmolecules.

[0087] By a “plurality of cells” or a “population of cells” herein ismeant roughly from about 10³ cells to 10⁸ or 10⁹, with from 10⁶ to 10⁸being preferred. This plurality of cells comprises a cellular library,wherein generally each cell within the library contains a member of theretroviral molecular library, i.e., a different candidate nucleic acid,although as will be appreciated by those in the art, some cells withinthe library may not contain a retrovirus, and some may contain more thanone. When methods other than retroviral infection are used to introducethe candidate nucleic acids into a plurality of cells, the distributionof candidate nucleic acids within the individual cell members of thecellular library may vary.

[0088] The methods rely on regulatable expression systems. Suitableregulatable expression systems for use in the methods of the presentevent are those having the following properties: a low level of basalexpression in the non-induced state; inducers that do not promotepleiotropic effects; high levels of expression in the induced state;highly specific induction of expression of a candidate nucleic acid ofinterest; and modulation of the level of induced expression. Examples ofregulatable expression systems having such properties include, but arenot limited to: a Tet inducible system (see e.g., Gossen et al. (1992)Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen et al. (1995) Science268:1766-1769); a FK506/rapamycin inducible system (see e.g., Spencer etal. (1993) Science 262:1019-1024; Belshaw et al. (1996) Proc. Natl.Acad. Sci. USA 93:4604-4607); a RU486/mifepristone inducible system; andan ecdysone inducible system (for review, see Rossi et al. (1989) Curr.Op. Biotech. 9:451-456).

[0089] In a preferred embodiment, a Tet inducible expression system isused in the methods of the present invention (see e.g., Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen et al. (1995)Science 268:1766-1769). In a Tet inducible expression system, aTet-dependent transactivator binds to a Tet operator (TetO) sequence andactivates the expression of a nucleic acid sequence operably linked tothe TetO sequence. Various Tet-dependent transactivators are known andeither bind to the operator sequence in the presence or the absence ofTet or an analogue thereof. Thus, Tet or an analogue thereof, acts as anagent mediating the binding of a transactivator to the operator sequenceand the expression of an operably linked nucleic acid sequence. Anexample of a tetracycline-dependent transactivator that binds to a TetOsequence in the presence of Tet, and not in the absence of Tet, is thereverse tetracycline-dependent transactivator (rtTA). An example of atetracycline-dependent transactivator that binds to the Tet) sequence inthe absence of Tet, and not in the presence of Tet, is thetetracycline-dependent transactivator (tTA).

[0090] In the methods of the present invention, the first elementregulates a second element that is operably linked to a nucleic acidsequence encoding a candidate bioactive agent. Preferably, the firstelement is a transactivator. In a preferred embodiment, the firstelement comprises the rtTA. In another preferred embodiment, the firstelement comprises the tTA. The first element can be expressed inducibly,constitutively, stably, transiently; or in trans or in cis relative to anucleic acid sequence that is operably linked to a second element. Inanother preferred embodiment, the transactivator is a polypeptide, andeven more preferably, the transactivator is a fusion protein. Thus, oneaspect of the invention relates to fusion proteins and nucleic acidsencoding fusion proteins. The term “fusion protein” as used hereinrefers to at least two polypeptides which are operably linked, eitherdirectly or indirectly, using a linker.

[0091] In a preferred embodiment, the transactivator fusion proteincomprises a first polypeptide that binds to a Tet operator sequence inthe presence of tetracycline (Tet) or an analogue thereof; and a secondpolypeptide that activates expression of a nucleic acid sequenceoperably linked to an operator sequence. The first polypeptide of thetransactivator fusion protein is preferably a mutated Tet repressor.

[0092] The wild-type Tet repressor (TetR) is a component of the E. colitetracycline (Tc) resistance system. Wild-type TetR binds to TetOsequences in the absence of Tet or an analogue thereof and repressesexpression of nucleic acid sequences operably linked to the TetOsequences (Gatz, C. et al. (1992) Plant J. 2:397-404).

[0093] The term “mutated TetR” or “mutant TetR” as used herein includespolypeptides having an amino acid sequence which is similar to awild-type TetR but which has at least one amino acid difference from thewild-type TetR. The term “wild-type TetR” as used herein describes anaturally occurring protein which represses transcription from TetOsequences in prokaryotic cells in the absence of Tet or an analoguethereof. The amino acid difference(s) between a mutated TetR and awild-type TetR may be a substitution of one or more amino acids,deletion of one or more amino acids, or addition of one or more aminoacids.

[0094] A suitable mutated TetR for use in the transactivator fusionproteins of the present invention binds to a TetO sequence, i.e., itretains the DNA binding specificity of a wild-type Tet repressor; andregulates expression in a reverse or opposite manner as compared to awild-type TetR, i.e., the mutated TetR binds to a TetO sequence only inthe presence of Tet or analogue thereof, rather than in the absence ofTet.

[0095] A mutated TetR having the functional properties described abovecan be constructed by substitution of amino acid residues in thesequence of a wild-type TetR. For example, a Tn10-derived TetR havingamino acid substitutions at amino acid positions 71, 95, 101 and 102 hasthe desired functional properties and thus can be used as the firstpolypeptide in the transactivator fusion protein of the invention. Theseand other amino acid substitutions, deletions or additions at these orother amino acid positions which retain the desired functionalproperties of the mutated TetR are known in the art (see, e.g., U.S.Pat. Ser. No. 6,136,954).

[0096] Further, the crystal structure of a TetR-Tet complex, asdescribed in Hinrichs, W. et al. (1994) Science 264:418-420, can be usedfor rational design of mutated Tet repressors. Amino acid positions 95,101 and 102 are located within the conserved Tet binding pocket. Thus,the Tet binding pocket of a TetR may mutated to generate a mutated TetRsuitable for inclusion in a transactivator fusion protein of the presentinvention.

[0097] Additional suitable mutated TetR can be constructed according tothe teachings of the invention and in the references cited herein. Anumber of different classes of TetR have been described, e.g., A, B, C,D and E (of which the Tn10-encoded repressor is a class B repressor).The amino acid sequences of the different classes of TetR share a highdegree of homology (i.e., 40-60% across the length of the proteins),including in the region encompassing the above-described mutations. Theamino acid sequences of various classes of TetR are described in Tovar,K. et al. (1988) Mol. Gen. Genet. 215:76-80. Accordingly, equivalentmutations to those described above for the Tn10-derived TetR can be madein other classes of TetR for inclusion in a transactivator fusionprotein of the invention. Suitable equivalent mutations will be apparentto those skilled in the art and can be constructed and tested forfunctionality by procedures described herein or in the cited references.Nucleotide and amino acid sequences of Tet repressors of the A, C, D andE classes are disclosed in Waters, S. H. et al. (1983) Nucl. Acids Res.11:6089-6105, Unger, B. et al. (1984) Nucleic acid 31:103-108, Unger, B.et al. (1984) Nucl Acids Res. 12:7693-7703 and Tovar, K. et al. (1988)Mol. Gen. Genet. 215:76-80, respectively. These wild-type TetR sequencescan be mutated according to the teachings herein and in the citedreferences, for inclusion in the transactivator fusion protein of thepresent invention. Additional suitable mutated Tet repressors (i.e.,having the desired functional properties described above) can beconstructed by mutagenesis of a wild-type TetR using methods known inthe art. The nucleotide and amino acid sequences of wild-type class BTet repressors are disclosed in Hillen, W. and Schollmeier, K. (1983)Nucl. Acids Res. 11:525-539 and Postle, K. et al. (1984) Nucl. AcidsRes. 12:4849-4863. The nucleotide and amino acid sequences of wild-typeclass A, C, D and E type repressors are cited above. A mutated TetR canbe created and selected, for example as follows: a nucleic acid (e.g.,DNA) encoding a wild-type TetR is subjected to random mutagenesis andthe resultant mutated nucleic acids are incorporated into an expressionvector and introduced into a host cell for screening. A screening assayis used which allows for selection of a TetR which binds to a Tetoperator sequence only in the presence of Tet or an analogue thereof.For example, a library of mutated nucleic acids in an expression vectorcan be introduced into an E. coli strain in which TetO sequences controlthe expression of a nucleic acid encoding a Lac repressor and the Lacrepressor controls the expression of a nucleic acid encoding anselectable marker (e.g., drug resistance). Binding of a TetR to TetOsequences in the bacteria will inhibit expression of the Lac repressor,thereby inducing expression of the selectable marker gene. Cellsexpressing the marker nucleic acid are selected based upon theselectable phenotype (e.g., drug resistance). For wild-type TetR,expression of the selectable marker nucleic acid will occur in theabsence of Tet. A nucleic acid encoding a mutated TetR is selected usingthis system based upon the ability of the nucleic acid to induceexpression of the selectable marker nucleic acid in the bacteria only inthe presence of Tet.

[0098] As mentioned above, the first polypeptide of the transactivatorfusion protein (e.g., the mutated TetR) has the property of bindingspecifically to a TetO sequence. Each class of TetR has a correspondingtarget TetO sequence. Accordingly, the term “Tet operator sequence” orATetO sequence” as used herein encompasses all classes of TetOsequences, e.g., class A, B, C, D, and E. Nucleotide sequences of thesefive classes of TetO sequences are described in Waters, S. H. et al.(1983) cited supra, Hillen, W. and Schollenmeier, K. (1983) cited supra,Stuber, D. and Bujard, H. (1981) Proc. Natl. Acad. Sci. USA 78:167-171,Unger, B. et al. (1984) cited supra and Tovar, K. et al. (1988) citedsupra. In a preferred embodiment, the mutated TetR is a Tn10-encodedrepressor (i.e., class B) and the TetO sequence is a class B Tetoperator sequence. Alternatively, a mutated class A TetR can be usedwith a class A TetO sequence, and so on for the other classes of TetRand TetO sequences.

[0099] Another approach for creating a mutated TetR which binds to aclass A Tet operator is to further mutate the already mutatedTn10-derived TetR described herein (a class B repressor) such that it nolonger binds efficiently to a class B operator but instead bindsefficiently to a class A operator as taught in the art (see, e.g.,Wissman et al. (1988) J. Mol. Biol. 202:397-406; Altschmied et al.(1988) EMBO J. 7:4011-4017). Accordingly, one can alter the bindingspecificity of the mutated Tn10-derived TetR as described herein byadditionally changing amino acid residue 40 from Thr to Ala by standardmolecular biology techniques (e.g., site directed mutagenesis).

[0100] A mutated TetR having specific mutations can be constructed byintroducing nucleotide changes into a nucleic acid encoding a wild-typerepressor by standard molecular biology techniques, e.g. site directedmutagenesis or PCR-mediated mutagenesis using oligonucleotide primersincorporating the nucleotide mutations. Alternatively, when a mutatedTetR is identified by selection from a library, the mutated nucleic acidcan be recovered from the library vector. To construct a transactivatorfusion protein suitable for use in the methods of the present invention,a nucleic acid encoding a mutated TetR is then ligated in-frame toanother nucleic acid encoding a transcriptional activation domain andthe fusion construct is incorporated into a recombinant expressionvector. The transactivator fusion protein can be expressed byintroducing the recombinant expression vector into a host cell.

[0101] The first polypeptide of the transactivator fusion protein isoperably linked to a second polypeptide. The second polypeptide directlyor indirectly activates expression in eukaryotic cells of a nucleic acidsequence operably linked to a TetO sequence. To operably link the firstand second polypeptides, the nucleic acid sequences encoding the firstand second polypeptides are ligated to each other in-frame to create achimeric nucleic acid encoding a transactivator fusion protein, althoughthe first and second polypeptides can be operably linked by other meansthat preserve the function of each polypeptide (e.g., chemicallycrosslinked). In a preferred embodiment, the second polypeptide of thetransactivator fusion protein itself possesses transcriptionalactivation activity (i.e., the second polypeptide directly activatesexpression). In another embodiment, the second polypeptide activatesexpression by an indirect mechanism, through recruitment of anactivation protein to interact with the fusion protein.

[0102] Polypeptides that function to directly or indirectly activategene expression in eukaryotic cells are well known in the art, and aresuitable for use in the construction of the second polypeptide of thetransactivator fusion protein. In particular, transcriptional activationdomains of many DNA binding proteins have been described and have beenshown to retain their activation function when the domain is transferredto a heterologous protein. A preferred polypeptide for use in thetransactivator fusion protein of the invention is the herpes simplexvirus virion protein 16 (referred to herein as VP16, the amino acidsequence of which is described in Triezenberg, S. J. et al. (1988) GenesDev. 2:718-729). In one embodiment, the second polypeptide of thetransactivator fusion protein comprises about 127 of the C-terminalamino acids of VP16 are used. In another embodiment, at least one copyof about 11 amino acids from the C-terminal region of VP16 which retaintranscriptional activation ability is used as the second polypeptide.Preferably, a dimer of this region (i.e., about 22 amino acids) is used.Suitable C-terminal peptide portions of VP16 are described in Seipel, K.et al. (EMBO J. (1992) 13:4961-4968).

[0103] Other polypeptides with the ability to activate expression ofnucleic acid sequences in eukaryotic cells can be used for constructionof the second polypeptide of the transactivator fusion protein of theinvention. Transcriptional activation domains found within variousproteins have been grouped into categories based upon similar structuralfeatures. Types of transcriptional activation domains include acidictranscription activation domains, proline-rich transcription activationdomains, serine/threonine-rich transcription activation domains andglutamine-rich transcription activation domains. Examples of acidictranscriptional activation domains include the VP16 regions alreadydescribed and amino acid residues 753-881 of GAL4. Examples ofproline-rich activation domains include amino acid residues 399-499 ofCTF/NF1 and amino acid residues 31-76 of AP2. Examples ofserine/threonine-rich transcription activation domains include aminoacid residues 1-427 of ITF1 and amino acid residues 2-451 of ITF2.Examples of glutamine-rich activation domains include amino acidresidues 175-269 of Oct1 and amino acid residues 132-243 of Sp1. Theamino acid sequences of each of the above described regions, and ofother useful transcriptional activation domains, are disclosed inSeipel, K. et al. (EMBO J. (1992) 13:4961-4968). In addition, novelactivation domains can be identified and constructed using methods knownin the art, and are within the scope of the invention.

[0104] In another embodiment, the second polypeptide of thetransactivator fusion protein indirectly activates transcription byrecruiting a transcriptional activator to interact with the fusionprotein. For example, a mutated tetR of the invention can be fused to apolypeptide domain (e.g., a dimerization domain) capable of mediating aprotein-protein interaction with a transcriptional activator protein,such as an endogenous activator present in a host cell. It has beendemonstrated that functional associations between DNA binding domainsand transactivation domains need not be covalent (see e.g., Fields andSong (1989) Nature 340:245-247; Chien et al. (1991) Proc. Natl. Acad.Sci. USA 88:9578-9582; Gyuris et al. (1993) Cell 75:791-803; and Zervos,A. S. (1993) Cell 72:223-232).

[0105] Accordingly, the second polypeptide of the fusion protein may notdirectly activate transcription but rather may form a stable interactionwith an endogenous polypeptide bearing a compatible protein-proteininteraction domain and transactivation domain. Examples of suitableinteraction (or dimerization) domains include leucine zippers(Landschulz et al. (1989) Science 243:1681-1688), helix-loop-helixdomains (Murre, C. et al. (1989) Cell 58:537-544) and zinc fingerdomains (Frankel, A. D. et al. (1988) Science 240:70-73). Interaction ofa dimerization domain present in the fusion protein with an endogenousnuclear factor results in recruitment of the transactivation domain ofthe nuclear factor to the fusion protein, and thereby to a Tet operatorsequence to which the fusion protein is bound.

[0106] A nucleic acid encoding a transactivator fusion protein of theinvention can be incorporated into a recombinant expression vector in aform suitable for expression of the fusion protein in a host cell. Thatis, the recombinant expression vector includes one or more regulatorysequences operably linked to the nucleic acid encoding the fusionprotein in a manner which allows for transcription of the nucleic acidinto mRNA and translation of the mRNA into the fusion protein. The term“regulatory sequence” is art-recognized and intended to includepromoters, enhancers and other expression control elements (e.g.,polyadenylation signals). Such regulatory sequences are known to thoseskilled in the art and are described in Goeddel, Nucleic acid ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). It should be understood that the design of the expression vectormay depend on such factors as the choice of the host cell to betransfected and/or the amount of fusion protein to be expressed.

[0107] When used in mammalian cells, a recombinant expression vector'scontrol functions are often provided by viral genetic material. Forexample, commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40. Use of viral regulatory elements todirect expression of the fusion protein can allow for high levelconstitutive expression of the fusion protein in a variety of hostcells. In a preferred recombinant expression vector, the sequencesencoding the fusion protein are flanked upstream (i.e., 5′) by the humancytomegalovirus IE promoter and downstream (i.e., 3′) by an SV40 poly(A)signal. The human cytomegalovirus IE promoter is described in Boshart etal. (1985) Cell 41:521-530. Other ubiquitously expressing promoterswhich can be used include the HSV-Tk promoter (disclosed in McKnight etal. (1984) Cell 37:253-262) and .beta.-actin promoters (e.g., the human-actin promoter as described by Ng et al. (1985) Mol. Cell. Biol.5:2720-2732).

[0108] Alternatively, the regulatory sequences of the recombinantexpression vector can direct expression of the fusion proteinpreferentially in a particular cell type, i.e., tissue-specificregulatory elements can be used. Non-limiting examples oftissue-specific promoters which can be used include the albumin promoter(liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).

[0109] Developmentally-regulated promoters are also encompassed, forexample the murine hox promoters (Kessel and Gruss (1990) Science249:374-379) and the -fetoprotein promoter (Campes and Tilghman (1989)Genes Dev. 3:537-546).

[0110] Alternatively, a self-regulating construct encoding atransactivator fusion protein can be constructed. To accomplish this,nucleic acid encoding the fusion protein is operably linked to a minimalpromoter sequence and at least one Tet operator sequence. When thisnucleic acid is introduced into a cell (e.g., in a recombinantexpression vector), a small amount of basal transcription of thetransactivator nucleic acid is likely to occur due to “leakiness”. Inthe presence of Tet or analogue thereof this small amount of thetransactivator fusion protein will bind to the Tet operator sequence(s)upstream of the candidate nucleic acid sequence encoding thetransactivator and stimulate additional transcription of the nucleicacid sequence encoding the transactivator, thereby leading to furtherproduction of the transactivator fusion protein in the cell.

[0111] It will be appreciated by those skilled in the art that such aself-regulating promoter can also be used in conjunction with othertetracycline-regulated transactivators, such as the wild-type Tetrepressor fusion protein (tTA) described in Gossen, M. and Bujard, H.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551, which binds to Tetoperators in the absence of Tet. When used in conjunction with thistransactivator, self-regulated transcription of the candidate nucleicacid sequence encoding this transactivator is stimulated in the absenceof Tet. The plasmid pUHD1 5-3, which comprises candidate nucleic acidsequences encoding the tTA described in Gossen and Bujard (1992), citedsupra, operably linked to a self-regulating promoter, has been depositedon Jul. 8, 1994 under the provisions of the Budapest Treaty at theDeutsche Sammlung Von Mikroorganismen und ZellKulturen GmbH (DSM) inBraunschweig, Germany and assigned deposit number DSM 9280.

[0112] In one embodiment, the recombinant expression vector containingthe transactivator fusion protein is a plasmid. Alternatively, therecombinant expression vector can be a virus, or portion thereof, whichallows for expression of a nucleic acid introduced into the viralnucleic acid. For example, replication defective retroviruses,adenoviruses and adeno-associated viruses can be used. Protocols forproducing recombinant retroviruses and for infecting cells in vitro orin vivo with such viruses can be found in Current Protocols in MolecularBiology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates,(1989), Sections 9.10-9.14 and other standard laboratory manuals.Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare well known to those skilled in the art. Examples of suitablepackaging virus lines include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am.The genome of adenovirus can be manipulated such that it encodes andexpresses a transactivator fusion protein but is inactivated in terms ofits ability to replicate in a normal lytic viral life cycle. See forexample Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al.(1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 eTet.) arewell known to those skilled in the art. Alternatively, anadeno-associated virus vector such as that described in Tratschin et al.(1985) Mol. Cell. Biol. 5:3251-3260 can be used to express atransactivator fusion protein.

[0113] The fusion nucleic acids of the present invention comprise asecond element that is regulatable by the first element. The secondelement is preferably an operator sequence, and more preferably a TetOsequence. In a preferred embodiment, the second element comprises anoligomer of a TetO sequence. The term “Tet operator sequence” as usedherein encompasses all classes of Tet operators (e.g., A, B, C, D andE). A nucleic acid sequence can be operably linked to a single TetOsequence, or for an enhanced range of regulation, it can be operablylinked to multiple TetO sequences (e.g., two, three, four, five, six,seven, eight, nine, ten or more operator sequences). In a preferredembodiment, the candidate nucleic acid sequence is operably linked toseven TetO sequences.

[0114] In a preferred embodiment, the transactivator fusion protein ofthe invention is used to regulate the expression of a nucleic acidsequence encoding a candidate bioactive agent. This nucleic acidsequence is operably linked to an operator sequence to which thetransactivator fusion protein binds. In a preferred embodiment, thetransactivator fusion protein regulates expression of a candidatenucleic acid sequence operably linked to at least one TetO sequence.Accordingly, another aspect of the invention relates to nucleic acidsequences that are operably linked to at least one TetO sequence. Suchnucleic acid sequences are also referred to herein as “tet-regulatedtranscription units” or “transcription units”. In a preferredembodiment, the transcription unit comprises a minimal promoter andcandidate nucleic acid sequence. The minimal promoter sequence isoperably linked to the candidate nucleic acid sequence in a 5′ to 3′direction by phosphodiester bonds. Accordingly, as used herein, theterms “candidate nucleic acid sequence” or “nucleic acid sequence”comprise a nucleic acid sequence encoding a candidate bioactive agentand may include operably linked functional elements. Examples offunctional elements, include but are not limited to, promoters (e.g.,minimal promoters), enhancers, restriction enzyme sites, and nucleicacid sequences encoding a peptide or an RNA.

[0115] The term “minimal promoter” as used herein refers to a partialpromoter sequence which defines the start site of transcription for anoperably linked nucleic acid sequence but which by itself is not capableof initiating transcription efficiently. Thus, the activity of such aminimal promoter is dependent upon the binding of a transcriptionalactivator to an operably linked operator sequence (e.g., one or moreTetO sequences). In one embodiment, the minimal promoter is from thehuman cytomegalovirus (as described in Boshart et al. (1985) Cell41:521-530). Preferably, nucleotide positions between about +75 to −53and +75 to −31 are used. Other suitable minimal promoters are known inthe art or can be identified by standard techniques.

[0116] Within a transcription unit, the candidate nucleic acid sequence(including an upstream minimal promoter sequence) is operably linked toat least one TetO sequence. For example, the TetO sequence(s) isoperably linked upstream or 5= of the minimal promoter sequence througha phosphodiester bond at a suitable distance to allow for transcriptionof the candidate nucleic acid sequence upon binding of a transactivator(e.g., rtTA or tTA) to the TetO sequence. That is, the transcriptionunit is comprised of, in a 5′ to 3′ direction: TetO sequence(s); aminimal promoter; and a candidate nucleic acid sequence. It will beappreciated by those skilled in the art that there is some flexibilityin the permissible distance between the TetO sequence(s) and the minimalpromoter, although typically the TetO sequences will be located withinabout 200-400 base pairs upstream of the minimal promoter. Suitablepromoter sequences are known in the art and described for example, inGossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA89:5547-5551.

[0117] Alternatively, since regulatory elements have been observed inthe art to function downstream of sequences to be transcribed, it islikely that the TetO sequence(s) can be operably linked downstream(i.e., 3′) of the candidate nucleic acid sequence. Thus, in thisconFiguration, the transcription unit is comprised of, in a 5′ to 3′direction: a minimal promoter; a candidate nucleic acid sequence; andTetO sequence(s). Again, it will be appreciated that there is likely tobe some flexibility in the permissible distance downstream at which theTetO sequence(s) can be linked.

[0118] A tet-regulated transcription unit can further be incorporatedinto a recombinant vector (e.g., a plasmid or viral vector) by standardrecombinant DNA techniques. The transcription unit, or recombinantvector in which it is contained, can be introduced into a host cell bystandard transfection techniques, such as those described herein. Itshould be appreciated that, after introduction of the transcription unitinto a population of host cells, it may be necessary to select a hostcell clone which exhibits low basal expression of the candidate nucleicacid sequence operably linked to the TetO sequence(s) (i.e., selectionfor a host cell in which the transcription unit has integrated at a sitethat results in low basal expression of the TetO-linked candidatenucleic acid sequence).

[0119] In one preferred embodiment, the candidate nucleic acid sequenceof the tet-regulated transcription unit encodes a candidate bioactiveagent that is a peptide, e.g., a cyclic peptide. Upon induction ofexpression of the candidate nucleic acid sequence and translation of theresultant mRNA, the peptide of interest is produced in a host cell. Inanother preferred embodiment, the candidate nucleic acid sequence of thetet-regulated transcription unit encodes a candidate bioactive agentthat is an RNA, e.g., an antisense RNA or ribozyme. Upon induction ofexpression of the candidate nucleic acid sequence, the RNA of interestis produced in the host cell.

[0120] Thus, examples of candidate bioactive agents include, but are notlimited to, nucleic acids and polypeptides. Further examples ofbioactive agents are cyclic peptides, RNA, antisense RNA, and DNA.Additional examples of nucleic acid sequences encoding a candidatebioactive agent, include but are not limited to, random nucleic acidsequences, and biased random nucleic acid sequences. Examples of nucleicacid sequences encoding a candidate bioactive agent, also include butare not limited to, full-length cDNA sequences, subsequences of afull-length cDNA, and antisense sequences of a full-length cDNA. Anotherexample of a nucleic acid sequence encoding a candidate bioactive agentis a nucleic acid sequence encoding an amino acid sequence that isin-frame or out-of-frame as compared to the open reading frame (ORF)encoded by the amino acid sequence of a full-length cDNA.

[0121] Alternatively, a transactivator of the invention can regulatetranscription of an endogenous nucleic acid sequence to which a TetOsequence(s) is operably linked. An “endogenous” nucleic acid sequence isa nucleic acid sequence which is present within the genome of a hostcell. An endogenous nucleic acid sequence can be operably linked to aTetO sequence(s) by homologous recombination between a TetO-containingrecombination vector and the endogeneous nucleic acid sequence. Forexample, a homologous recombination vector can be prepared whichincludes at least one TetO sequence and a miminal promoter sequenceflanked at the 3′ end by sequences representing the coding region of theendogenous nucleic acid sequence and flanked at the 5′ end by sequencesfrom the upstream region of the endogenous nucleic acid sequence byexcluding the actual promoter region of the endogenous nucleic acidsequence. The flanking sequences are of sufficient length for successfulhomologous recombination of the vector DNA with the endogenous gene.Preferably, upon homologous recombination between the vector DNA and theendogenous nucleic acid sequence in a host cell, a region of theendogenous promoter is replaced by the vector DNA containing one or moreTetO sequences operably linked to a minimal promoter. Thus, expressionof the endogenous nucleic acid sequence is no longer under the controlof its endogenous promoter but rather is placed under the control of theTetO sequence(s) and the minimal promoter.

[0122] In another embodiment, TetO sequences can be inserted elsewherewithin an endogenous gene, preferably within a 5′ or 3′ regulatoryregion, via homologous recombination to create an endogenous nucleicacid whose expression can be regulated by a tet. An endogenous nucleicacid sequence having TetO sequences inserted into a non-criticalregulatory region will retain the ability to be expressed in a normalconstitutive and/or tissue-specific manner but, additionally, can bedownregulated by a tetracycline-controlled transcriptional inhibitorprotein (described infra) in a controlled manner. For example,constitutive expression of such a modified endogenous nucleic acidsequence can be inhibited by in the presence of Tet or analogue thereofusing an inhibitor fusion protein that binds to TetO sequences in thepresence of Tet or analogue thereof.

[0123] In the present invention, the expression of a candidate nucleicacid operably linked to a second element (e.g., an operator sequence)that is regulatable by a first element (e.g., a transactivator). Thus,these components, the first element and the candidate nucleic acidoperably linked to an operator sequence, are present in a host cell, andcan be contained on the same nucleic acid or separate nucleic acids. Thepresence of these components in the same host cell can be achieved in anumber of different ways. For example, a host cell can be transfectedwith a first nucleic acid encoding, e.g., a first element, stablytransfected cells can be selected, and then the transfected cells can bere-transfected (also referred to as “supertransfected”) with a secondnucleic acid comprising, e.g., the second element operably linked to acandidate nucleic acid. Two distinct selectable markers can be used forselection, e.g., uptake of the first nucleic acid can be selected withG418 and uptake of the second nucleic acid can be selected withhygromycin. Alternatively, a single population of cells can betransfected with nucleic acid corresponding to both components of thesystem.

[0124] Accordingly, in one aspect, the invention provides a firstnucleic acid encoding a transactivator fusion protein and a secondnucleic acid comprising a candidate nucleic acid sequence operablylinked to at least one TetO sequence. The transactivator of the firstnucleic acid comprises a first polypeptide and second polypeptide. Thefirst polypeptide binds to a TetO sequence in the presence of Tet oranalogue thereof and is operably linked to a second polypeptide whichactivates transcription in eukaryotic cells.

[0125] In one embodiment, the two nucleic acids are two separatemolecules (e.g., two different vectors). Thus, in this embodiment a hostcell is cotransfected with the two nucleic acid molecules orsuccessively transfected first with one nucleic acid molecule and thenthe other nucleic acid molecule. In another embodiment, the two nucleicacids are linked (i.e., colinear) in the same nucleic acid molecule(e.g., a single vector). Thus, in this embodiment a host cell istransfected with the single nucleic acid molecule. Further, the hostcell may be a cell cultured in vitro or a cell present in vivo.

[0126] The third element of the present invention, induces or repressesthe expression of a candidate nucleic acid. In a preferred embodiment,the third element is an agent which induces expression of a candidatenucleic acid, e.g., by binding to a transactivator (e.g., rtTA). Inanother preferred embodiment, the third element is an agent whichrepresses expression of a candidate nucleic acid, e.g., by binding to atransactivator (e.g., tTA). In a preferred embodiment, the agentcomprises tetracycline or an analogue thereof, e.g., doxycycline (Dox).

[0127] The term “tetracycline analogue” is intended to include compoundswhich are structurally related to Tet and which bind to the TetR with aK_(a) of at least about 10⁶ M⁻¹. Preferably, the Tet analogue binds withan affinity of about 10⁹ M⁻¹ or greater. Examples of such Tet analoguesinclude, but are not limited to, anhydrotetracycline, Dox,chlorotetracycline, oxytetracycline and others disclosed by Hlavka andBoothe, “The Tetracyclines,” in Handbook of Experimental Pharmacology78, R. K. Blackwood et al. (eds.), Springer-Verlag, Berlin-New York,1985; L. A. Mitscher, “The Chemistry of the Tetracycline Antibiotics”,Medicinal Research 9, Dekker, N.Y., 1978; Noyee Development Corporation,“Tetracycline Manufacturing Processes” Chemical Process Reviews, ParkRidge, N.J., 2 volumes, 1969; R. C. Evans, “The Technology of theTetracyclines”, Biochemical Reference Series 1, Quadrangle Press, NewYork, 1968; and H. F. Dowling, “Tetracycline”, Antibiotic Monographs,no. 3, Medical Encyclopedia, New York, 1955. Preferred Tet analogues forhigh level stimulation of transcription are anhydrotetracycline and Dox.A Tet analogue can be chosen which has reduced antibiotic activitycompared to Tet. Examples of such Tet analogues are anhydrotetracycline,epioxytetracycline and cyanotetracycline.

[0128] In a preferred embodiment, to induce or repress nucleic acidexpression in a cell in vitro, the cell is contacted with Tet or aanalogue thereof by culturing the cell in a medium containing thecompound. Preferably, the concentration range of the Tet or analoguethereof in the cell medium is between about 10 and about 1000 ng/ml. Tetor analogue thereof can be directly added to media in which cells arealready being cultured, or more preferably for high levels of nucleicacid induction. Additionally, the cells can be harvested from Tet-freemedia and cultured in fresh media containing Tet, or an analoguethereof, or vice versa.

[0129] The use of different Tet analogues allows for the modulation ofthe level of expression of a nucleic acid sequence operably linked to aTetO sequence, for example, by adjusting the concentration of Tet or ananalogue thereof in contact with the cells. For example,anhydrotetracycline and doxycycline are known to be strong inducers ofexpression, e.g., in a system where a transactivator binds to a TetOsequence in the presence of Tet or analogue thereof. From the uninducedstate to the induced state, the increase in expression of a nucleic acidoperably linked to a TetO sequence is typically about 1000- to2000-fold, and can be at least about 20,000 fold. In the samesystem,Tet, chlorotetracycline and oxytetracycline have been found to beweaker inducers of expression, i.e., from the uninducec state to theinduced state, the increase in expression at least about 10-fold. Thus,an appropriate Tet analogue can be selected, for use in the methods ofthe present invention, based upon the desired level of induction orrepression of expression of nucleic acid sequence operably linked to aTetO sequence. It is also possible to change the level of expressionover time, in a host cell, of a nucleic acid operably linked to a TetOsequence, by changing the Tet analogue used to induce or repressexpression. For example, in some situations it may be desirable to havea strong burst of expression initially and then have a sustained lowerlevel of expression.

[0130] Accordingly, a first agent (e.g., a first Tet analogue) whichstimulates a high levels of expression can be used initially as thethird element in the methods of the present invention and then the thirdelement can be switched to a second agent (e.g., a second analogue)which stimulates a lower level of expression. Moreover, when regulatingthe expression of multiple nucleic acid sequences (e.g., when onesequence is regulated by a one of class TetO sequence(s) and the otheris regulated by another class of TetO sequence(s), it may be possible toindependently vary the level of expression of each sequence dependingupon which transactivator fusion protein is used to regulatetranscription and which Tet analogue is used.

[0131] In a preferred embodiment, a host cell comprises an rtTA and afusion nucleic acid comprising a nucleic acid sequence operably linkedto a TetO sequence, and a high level expression of the nucleic acidsequence does not occur in the absence of a third element, for example,tetracycline or analogues thereof. The level of basal expression of thecandidate nucleic acid sequence may vary depending upon the host celland site of integration of the sequence, but is generally quite low oreven undetectable in the absence of Tet. In order to induce expression,the host cell is contacted with Tet or analogue thereof. In order torepress expression, the contacting of Tet or analogue thereof with thecells is modulated. In a preferred embodiment, the contacting ismodulated by adjusting the concentration of the Tet or analogue thereof,for example, in the cell medium. Accordingly, another aspect of theinvention relates to methods for inducing or repressing expression of anucleic acid sequence operably linked to a TetO sequence in a host cellwhich expresses a transactivator of the invention, by modulating thecontacting of Tet or an analogue thereof with the host cell.

[0132] In a preferred embodiment, a host cell comprises an tTA and afusion nucleic acid comprising a candidate nucleic acid sequenceoperably linked to a TetO sequence, high level expression of thecandidate nucleic acid sequence occurs only in the presence of the rtTAand, expression is repressed in the presence of Tet or an analoguethereof. The level of basal expression of the candidate nucleic acidsequence may vary depending upon the host cell and site of integrationof the sequence, but is generally quite low or even undetectable in theabsence of tTA. In order to induce expression, tTA is expressed in thehost cell. In order to repress expression, the cells are contacted withTet or an analogue thereof. In a preferred embodiment, the contacting ismodulated by adjusting the concentration of the Tet or analogue thereof,for example, in the cell medium. Accordingly, another aspect of theinvention relates to methods for inducing or repressing expression of anucleic acid sequence operably linked to a TetO sequence in a host cellcomprising a transactivator of the invention, by modulating thecontacting of Tet or an analogue thereof with the host cell.

[0133] Different transactivator fusion proteins are likely to exhibitdifferent levels of responsiveness to Tet analogues. Thus, the level ofinduction or repression of expression by a particular combination oftransactivator fusion protein and Tet analogue can be determined bytechniques described herein or known in the art. Additionally, the levelof expression can be modulated by varying the concentration of the Tetanalogue. Thus, in the methods of the present invention, expression of anucleic acid operably linked to a TetO sequence can be regulated byturning the expression on or off, but also by modulating the level ofexpression at intermediate levels (between induction and repression)depending on the type and concentration of the Tet analogue used in themethods.

[0134] Another aspect of the invention relates to inhibitor fusionproteins. The inhibitor fusion proteins are constructed similarly to thetransactivator fusion proteins in that the fusion proteins comprise afirst and second polypeptide, and the first polypeptide is a mutatedTetR. However, in contrast to the transactivator fusion protein, thesecond polypeptide of the inhibitor fusion protein has a domain thatinhibits expression (rather than activates expression as in thetransactivator fusion protein) in eukaryotic cell of a nucleic acidsequence operably linked to a TetO sequence. Thus, inhibitor fusionproteins can be used to downregulate the expression of a nucleic acidsequence operably linked to a TetO sequence, and can be used in themethods of the present invention to screen for an altered cellularphenotype as described herein. For example, the level of basal,constitutive expression of a nucleic acid sequence operably linked to anoperator sequence may vary depending upon the type of cell in which thenucleic acid is introduced or the site of integration of the nucleicacid. Therefore, the inhibitor fusion proteins of the invention can, ina controlled manner, inhibit the expression of a nucleic acid operablylinked to an operator sequence.

[0135] In one embodiment, the inhibitor fusion protein comprises a firstpolypeptide that binds to Tet operator sequences in the absence, but notthe presence, of Tet or an analogue thereof, operably linked to aheterologous second polypeptide that inhibits transcription ineukaryotic cells. In another embodiment, the inhibitor fusion proteincomprises a first polypeptide that binds to a Tet operator sequence inthe presence, but not the absence, of Tet or analogue thereof, operablylinked to a heterologous second polypeptide that inhibits transcriptionin eukaryotic cells. The term “heterologous” is intended to mean thatthe second polypeptide is derived from a different protein than thefirst polypeptide. Like the transactivator fusion proteins, theinhibitor fusion proteins can be prepared using standard recombinant DNAtechniques as described herein.

[0136] Proteins and polypeptide domains within proteins which canfunction to inhibit transcription in eukaryotic cells have beendescribed in the art (for reviews see, e.g., Renkawitz, R. (1990) Trendsin Genetics 6:192-197; and Herschbach, B. M. and Johnson, A. D. (1993)Annu. Rev. Cell. Biol. 9:479-509) have suitable inhibitor domains foruse in the inhibitor fusion proteins of the present invention. Suchtranscriptional inhibitor domains have been referred to in the art as“silencing domains” or “repressor domains.” Although the precisemechanism by which many of these polypeptide domains inhibittranscription is not known (and the invention is not intended to belimited by mechanism), there are several possible means by whichrepressor domains may inhibit transcription, including: competitiveinhibition of binding of either activator proteins or the generaltranscriptional machinery; prevention of the activity of a DNA boundactivator; and negative interference with the assembly of a functionalpreinitiation complex of the general transcription machinery. Thus, aninhibitor domain may have a direct inhibitory effect on thetranscriptional machinery or may inhibit transcription indirectly byinhibiting the activity of activator proteins.

[0137] Accordingly, polypeptide containing an inhibitor domain can acteither directly or indirectly to inhibit expression. As used herein a“reduction” in the level of expression of a nucleic acid sequenceoperably linked to an operator sequence refers to a diminution in thelevel or amount of expression of the nucleic acid compared to the levelor amount prior to regulation by the transcriptional inhibitor protein.Transcriptional inhibition may be partial or complete. The terms“silencer”, “repressor” and “inhibitor” are used interchangeably hereinto describe an inhibitor protein, or domains thereof, that can inhibitexpression of a nucleic acid sequence operably linked to an operatorsequence.

[0138] A “repressor” or “silencer” domain as used herein is apolypeptide domain that retains its repressor function (e.g., repressionof expression of a nucleic acid sequence operably linked to an operatorsequence) when the domain is transferred to a heterologous protein.Proteins which have been demonstrated to have repressor domains that canfunction when transferred to a heterologous protein include the v-erbAonconucleic acid product (Baniahmad, A. et al. (1992) EMBO J.11:1015-1023), the thyroid hormone receptor (Baniahmad, supra), theretinoic acid receptor (Baniahmad, supra), and the Drosophila Krueppel(Kr) protein (Licht, J. D. et al. (1990) Nature 346:76-79; Sauer, F. andJackle, H. (1991) Nature 353:563-566; Licht, J. D. et al. (1994) Mol.Cell. Biol. 14:4057-4066). Non-limiting examples of other proteins whichhave transcriptional repressor activity in eukaryotic cells include theDrosophila homeodomain protein even-skipped (eve), the S. cerevisiaeSsn6/Tup1 protein complex (see Herschbach and Johnson, supra), the yeastSIR1 protein (see Chien, et al. (1993) Cell 75:531-541), NeP1 (seeKohne, et al. (1993) J. Mol. Biol. 232:747-755), the Drosophila dorsalprotein (see Kirov, et al. (1994) Mol. Cell. Biol. 14:713-722; Jiang, etal. (1993) EMBO J. 12:3201-3209), TSF3 (see Chen, et. al. (1993) Mol.Cell. Biol. 13:831-840), SF1 (see Targa, et al. (1992) Biochem. Biophys.Res. Comm. 188:416-423), the Drosophila hunchback protein (see Zhang, etal. (1992) Proc. Natl. Acad. Sci. USA 89:7511-7515), the Drosophilaknirps protein (see Gerwin, et al. (1994) Mol. Cell. Biol.14:7899-7908), the WT1 protein (Wilm's tumor nucleic acid product) (seeAnant, et al. (1994) Onconucleic acid 9:3113-3126; Madden et al., (1993)Onconucleic acid 8:1713-1720), Oct-2.1 (see Lillycrop, et al. (1994)Mol. Cell. Biol. 14:7633-7642), the Drosophila engrailed protein (seeBadiani, et al. (1994) Genes Dev. 8:770-782; Han and Manley, (1993) EMBOJ. 12:2723-2733), E4BP4 (see Cowell and Hurst, (1994) Nucleic Acids Res.22:59-65) and ZF5 (see Numoto, et al. (1993) Nucleic Acids Res.21:3767-3775),

[0139] In additional aspects described below, the invention providesalternative approaches to regulating the expression of one or morenucleic acid sequences. These alternative approaches to regulatingexpression of nucleic acid sequences operably linked to TetO sequencesare suitable for use in the methods of the present invention to screenfor cells having an altered phenotype due to the presence of a candidatebioactive agent.

[0140] In addition to regulating nucleic acid expression using either atransactivator fusion protein or inhibitor fusion protein alone, thesetwo types of proteins, can be used in combination to allow for bothpositive and negative regulation of expression of one or more nucleicacids in a host cell. Positive regulation of expression refers toinduction or increase in expression, whereas negative regulation ofexpression refers to repression or reduction in expression. Thus, aninhibitor protein that binds to TetO either 1) in the absence, but notthe presence, of Tet; or 2) in the presence, but not the absence, of Tetor analogue thereof, can be used in combination with a transactivatorfusion protein that binds to TetO either 1 in the absence, but not thepresence, of Tet or analogue thereof; or 2) in the presence, but not theabsence, of Tet or analogue thereof. Transactivator proteins that bindto TetO in the absence, but not the presence, of Tet or analogue thereofare described herein, and in U.S. Pat. No. 5,464,758, U.S. Ser. No.08/076,327 and U.S. Pat. No. 5,650,298. Transactivator proteins thatbind to TetO in the presence, but not the absence, of Tet are describedherein, and in U.S. Ser. No. 08/270,637 and U.S. Pat. No. 5,654,168.

[0141] When more than one TetR-containing fusion protein is expressed ina host cell, additional steps may be taken to inhibit heterodimerizationbetween the different TetR-containing fusion proteins. For example, atransactivator composed of a TetR of one class may be used incombination with an inhibitor fusion protein composed of a TetR of asecond, different class that does not heterodimerize with the firstclass of TetR. Alternatively, amino acid residues of the TetR involvedin dimerization may be mutated to inhibit heterodimerization. However,even if some heterodimerization between transactivator and inhibitorfusion proteins occurs in a host cell, sufficient amounts of homodimersshould be produced to allow for efficient positive and negativeregulation as described herein.

[0142] It will be appreciated by those skilled in the art that variouscombinations of activator and inhibitor proteins can be used to regulatea single nucleic acid sequence operably linked to a TetO sequence inboth a positive and negative manner or to regulate multiple nucleic acidsequences operably linked to TetO sequences in a coordinated manner orin an independent manner using the teachings described herein or knownin the art. Several non-limiting examples of how the transactivator andinhibitor fusion proteins may be used in combination are describedfurther below. However, many other possible combinations will be evidentto the skilled artisan in view of the teachings herein and known in theart.

[0143] In one embodiment expression of a nucleic acid acid sequenceoperably linked to a TetO sequence in a host cell is regulated in both anegative and positive manner by the combination of an inhibitor fusionprotein that binds to TetO in the absence, but not the presence, of Tetor analogue thereof (referred to as a Tet controlled silencing domain,or tSD) and an transactivator fusion protein that binds to TetO in thepresence, but not the absence, of Tet or analogue thereof (e.g., rtTA).In addition to TetO sequences, the nucleic acid sequence is operablylinked to a promoter, and may contain other positive regulatory elements(e.g., enhancer sequences) that contribute to basal level, constitutiveexpression of the nucleic acid in the host cell. Binding of tSD to theTetO sequences in the absence of Tet or analogue thereof inhibits thebasal constitutive expression of the nucleic acid sequence, thus keepingthe expression of the nucleic acid sequence in a repressed or uninducedstate until expression is desired. When expression is desired, theconcentration of Tet or analogue thereof in contact with the host cellis increased. Upon contacting a host cell with Tet or an analoguethereof, tSD loses the ability to bind to TetO sequences whereas thepreviously unbound rtTA acquires the ability to bind to TetO sequences.The resultant binding of rtTA to the TetO sequences operably linked to anucleic acid sequence of interest thus induces expression of the nucleicacid sequence. The level of expression may be controlled by theconcentration of Tet or analogue thereof, type of Tet analogue, durationof induction, and type of rtTA (e.g., class of TetR and transactivatordomain, as described previously herein). It will be appreciated that thecombination of transactivator and inhibitor fusion proteins (i.e., wherethe inhibitor binds in the presence but not the absence of Tet oranalogue thereof, and the transactivator binds in the absence but notthe presence of Tet or analogue thereof) can also be used in the methodsof the present invention. In this case, expression of the nucleic acidsequence of interest is repressed by contacting the host cell with Tet(e.g., culture with Tet or analogue) and nucleic acid expression isactivated by removal of the drug.

[0144] In another embodiment, the activator and inhibitor fusionproteins are used in combination to coordinately regulate, in both apositive and negative manner, two nucleic acid sequences operably linkedto a TetO sequence but are expressed bidirectionally with respect toeach other. In this case, a first nucleic acid and a second nucleic acidare linked to the same TetO sequence(s), but in opposite orientations.The inhibitor fusion protein is used to repress basal levels ofexpression of both the first nucleic acid and the second nucleic acid ina coordinate manner, whereas the transactivator fusion protein is usedto stimulate expression of the first nucleic acid and the second nucleicacid in a coordinate manner.

[0145] In another embodiment, the activator and inhibitor fusionproteins are used to independently regulate two or more nucleic acidsequences each operably linked to their respective TetO sequence, whereeach TetO sequence is of a different class. In one embodiment, atransactivator fusion protein that binds to one class of TetO sequences(e.g., class A) in the presence, but not the absence of Tet or analogueis used in combination with an inhibitor fusion protein that binds to asecond, different class of TetO sequences (e.g., class B) also in thepresence, but not the absence, of Tet or analogue. For example, a hostcell containing a first nucleic acid sequence operably linked to class ATetO sequences and second nucleic acid sequence operably linked to classB TetO sequences, both nucleic acid sequences will be expressed at basallevels in the absence of Tet or an analogue thereof, whereas expressionof the first nucleic acid will be stimulated in the presence of Tet oran analogue thereof and expression of the second nucleic acid will berepressed in the presence of Tet or analogue thereof.

[0146] Alternatively, in another embodiment, the transactivator fusionprotein binds to one class of TetO sequences (e.g., class A) in thepresence, but not the absence, of Tet or analogue thereof and theinhibitor fusion protein binds to a second, different class of TetOsequences (e.g., class B) in the absence but not the presence of Tet oranalogue. For example, in the host cell, the first nucleic acid sequencewill be expressed at basal levels in the absence of Tet or an analoguethereof and will be stimulated in the presence of Tet or an analoguethereof, whereas the expression of the second nucleic acid sequence willbe repressed in the absence of the Tet or an analogue thereof but willhave basal levels expression in the presence of Tet or analogue thereof.Various other possible combinations will be apparent to the skilledartisan.

[0147] By “fusion nucleic acid” or grammatical equivalents thereof ismeant a nucleic acid comprising functional elements that may or may notbe operably linked. Examples of functional elements include, but are notlimited to, a promoter, enhancer, operator sequence, restriction enzymesite, candidate nucleic acid, and nucleic acid encoding a polypeptide orRNA. In a preferred embodiment the fusion nucleic acid comprises asecond element and a candidate nucleic acid. In another preferredembodiment, the fusion nucleic acid comprises a nucleic acid encoding afirst element. As outlined herein, fusion nucleic acids, or othercomponents of the system such as fusion partners as well as the vectorsthemselves, can include reporter proteins.

[0148] By “candidate nucleic acid” or grammatical equivalents thereof ismeant a nucleic acid comprising a nucleic acid sequence encoding acandidate bioactive agent. The candidate nucleic acid of the presentinvention can also comprises functional elements, e.g., a promoter,enhancer, and restriction enzyme site. In another preferred embodiment,the candidate nucleic acid comprises and minimal promoter operablylinked to a nucleic acid sequence encoding the bioactive agent.

[0149] By “candidate nucleic acid sequence” or grammatical equivalentsthereof is meant a nucleic acid sequence comprising a nucleic acidsequence encoding a candidate bioactive agent.

[0150] By “candidate bioactive agents” or “candidate drugs” or“candidate expression products” or grammatical equivalents herein ismeant the expression product of a candidate nucleic acid sequence whichmay be tested for the ability to alter the phenotype of a cell. As isdescribed below, the candidate bioactive agents are the expressionproducts of a candidate nucleic acid sequence, and encompass severalchemical classes, including peptides and nucleic acids such as DNA,cDNA, messenger RNA (mRNA), antisense RNA, and ribozyme components.Thus, the candidate bioactive agents (expression products) may be eithertranslation products of the candidate nucleic acid sequence, i.e.,peptides, or transcription products of the candidate nucleic acidsequences, i.e., either DNA or RNA.

[0151] In a preferred embodiment, the candidate bioactive agents aretranslation products of candidate nucleic acid sequences. In thisembodiment, a library of fusion nucleic acids each comprising acandidate nucleic acid sequence are introduced into the cells, and thecells express the nucleic acid sequence to form peptides. Thus, in thisembodiment, the candidate bioactive agents are peptides. Generally,peptides ranging from about 4 amino acids in length to about 100 aminoacids may be used, with peptides ranging from about 5 to about 50 beingpreferred, with from about 5 to about 30 being particularly preferredand from about 6 to about 20 being especially preferred.

[0152] In a preferred embodiment, the candidate bioactive agents aretranscription products of candidate nucleic acid sequences and, thus,the candidate bioactive agents are also nucleic acids. The transcriptionproducts may be either primary transcripts or secondary translationproducts. That is, using the retroviral reverse transcriptase, primaryDNA is made which is later converted into double stranded DNA.Additionally, using the primary DNA, RNA transcripts can be generatedwithin the cell, including mRNA, antisense RNA and ribozymes or portionsthereof.

[0153] At a minimum, the candidate bioactive agents comprise randomizedexpression products of the nucleic acid sequence of the fusion nucleicacids. That is, every candidate bioactive agent has a randomizedportion, as defined below, that is the basis of the screening methodsoutlined herein. In addition, to the randomized portion, the candidatebioactive agent may also include a fusion partner.

[0154] In a preferred embodiment, the candidate bioactive agents arelinked to a fusion partner. By “fusion partner” or “functional group”herein is meant a sequence that is associated with the candidatebioactive agent, that confers upon all members of the library in thatclass a common function or ability. Fusion partners can be heterologous(i.e., not native to the host cell), or synthetic (not native to anycell). Suitable fusion partners include, but are not limited to: a)presentation structures, as defined below, which provide the candidatebioactive agents in a conformationally restricted or stable form; b)targeting sequences, defined below, which allow the localization of thecandidate bioactive agent into a subcellular or extracellularcompartment; c) rescue sequences as defined below, which allow thepurification or isolation of either the candidate bioactive agents orthe nucleic acids encoding them; d) stability sequences, which conferstability or protection from degradation to the candidate bioactiveagent or the nucleic acid encoding it, for example resistance toproteolytic degradation; e) dimerization sequences, to allow for peptidedimerization; or f) any combination of a), b), c), d), and e), as wellas linker sequences as needed.

[0155] In a preferred embodiment, the fusion partner is a presentationstructure. By “resentation structure” or grammatical equivalents hereinis meant a sequence, which, when fused to candidate bioactive agents,causes the candidate agents to assume a conformationally restrictedform. Proteins interact with each other largely through conformationallyconstrained domains. Although small peptides with freely rotating aminoand carboxyl termini can have potent functions as is known in the art,the conversion of such peptide structures into pharmacological agents isdifficult due to the inability to predict side-chain positions forpeptidomimetic synthesis. Therefore the presentation of peptides inconformationally constrained structures will benefit both the latergeneration of pharmaceuticals and will also likely lead to higheraffinity interactions of the peptide with the target protein. This facthas been recognized in the combinatorial library generation systemsusing biologically generated short peptides in bacterial phage systems.A number of workers have constructed small domain molecules in which onemight present randomized peptide structures.

[0156] While the candidate bioactive agents may be either nucleic acidor peptides, presentation structures are preferably used with peptidecandidate agents. Thus, synthetic presentation structures, i.e.,artificial polypeptides, are capable of presenting a randomized peptideas a conformationally-restricted domain. Generally such presentationstructures comprise a first portion joined to the N-terminal end of therandomized peptide, and a second portion joined to the C-terminal end ofthe peptide; that is, the peptide is inserted into the presentationstructure, although variations may be made, as outlined below. Toincrease the functional isolation of the randomized expression product,the presentation structures are selected or designed to have minimalbiologically activity when expressed in the target cell.

[0157] Preferred presentation structures maximize accessibility to thepeptide by presenting it on an exterior loop. Accordingly, suitablepresentation structures include, but are not limited to, minibodystructures, loops on -sheet turns and coiled-coil stem structures inwhich residues not critical to structure are randomized, zinc-fingerdomains, cysteine-linked (disulfide) structures, transglutaminase linkedstructures, cyclic peptides, B-loop structures, helical barrels orbundles, leucine zipper motifs, etc.

[0158] In a preferred embodiment, the presentation structure is acoiled-coil structure, allowing the presentation of the randomizedpeptide on an exterior loop. See, for example, Myszka et al., Biochem.33:2362-2373 (1994), hereby incorporated by reference, and FIG. 28).Using this system investigators have isolated peptides capable of highaffinity interaction with the appropriate target. In general,coiled-coil structures allow for between 6 to 20 randomized positions.

[0159] A preferred coiled-coil presentation structure is as follows:MGCAALESEVSALESEVASLESEVAALGRGDMPLAAVKSKLSAVKSKLASVKSKLAACGPP. Theunderlined regions represent a coiled-coil leucine zipper region definedpreviously (see Martin et al., EMBO J. 13(22):5303-5309 (1994),incorporated by reference). The bolded GRGDMP region represents the loopstructure and when appropriately replaced with randomized peptides(i.e., candidate bioactive agents, generally depicted herein as (X)_(n),where X is an amino acid residue and n is an integer of at least 5 or 6)can be of variable length. The replacement of the bolded region isfacilitated by encoding restriction endonuclease sites in the underlinedregions, which allows the direct incorporation of randomizedoligonucleotides at these positions. For example, a preferred embodimentgenerates a Xhol site at the double underlined LE site and a HindIIIsite at the double-underlined KL site.

[0160] In a preferred embodiment, the presentation structure is aminibody structure. A “inibody” is essentially composed of a minimalantibody complementarity region. The minibody presentation structuregenerally provides two randomizing regions that in the folded proteinare presented along a single face of the tertiary structure. See forexample Bianchi et al., J. Mol. Biol. 236(2):649-59 (1994), andreferences cited therein, all of which are incorporated by reference).Investigators have shown this minimal domain is stable in solution andhave used phage selection systems in combinatorial libraries to selectminibodies with peptide regions exhibiting high affinity, Kd=10⁻⁷, forthe pro-inflammatory cytokine IL-6.

[0161] A preferred minibody presentation structure is as follows:MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSRDTSQSILYLQKKKG PP.The bold, underline regions are the regions which may be randomized. Theitalicized phenylalanine must be invariant in the first randomizingregion. The entire peptide is cloned in a three-oligonucleotidevariation of the coiled-coil embodiment, thus allowing two differentrandomizing regions to be incorporated simultaneously. This embodimentutilizes non-palindromic BstXl sites on the termini.

[0162] In a preferred embodiment, the presentation structure is asequence that contains generally two cysteine residues, such that adisulfide bond may be formed, resulting in a conformationallyconstrained sequence. This embodiment is particularly preferred whensecretory targeting sequences are used. As will be appreciated by thosein the art, any number of random sequences, with or without spacer orlinking sequences, may be flanked with cysteine residues. In otherembodiments, effective presentation structures may be generated by therandom regions themselves. For example, the random regions may be “oped”with cysteine residues which, under the appropriate reDox conditions,may result in highly crosslinked structured conformations, similar to apresentation structure. Similarly, the randomization regions may becontrolled to contain a certain number of residues to confer β-sheet or-helical structures.

[0163] In a preferred embodiment, the presentation structure is areporter protein. In a preferred embodiment, the reporter protein can beused as a direct label, for example a detection protein for sorting thecells or for cell enrichment by FACS. In this embodiment, the proteinproduct of the reporter gene itself can serve to distinguish cells thatare expressing the reporter gene. In this embodiment, suitable reportergenes include those encoding green fluorescent protein (GFP; Chalfie, M.et al. (1994) Science 263:802-05; and EGFP; Clontech—Genbank AccessionNumber U55762), blue fluorescent protein (BFP; Quantum Biotechnologies,Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) CanadaH3H 1J9; Stauber, R. H. (1998) Biotechniques 24:462-71; Heim, R. et al.(1996) Curr. Biol. 6:178-82), enhanced yellow fluorescent protein (EYFP;1. Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto,Calif. 94303),, Renilla reniformis GFP (WO 99/49019), Ptilosarcusgurneyi GFP (WO 99/49019; U.S. Ser. No. 60/164,592; U.S. Ser. No.09/710,058; U.S. Ser. No. 60/290,287), Renilla mulleris GFP (WO99/49019; U.S. Ser. No. 60/164,592; U.S. Ser. No. 09/710,058; U.S. Ser.No. 60/290,287), luciferases (for example, firefly, Kennedy, H. J. etal. (1999) J. Biol. Chem. 274:13281-91; Renilla reniformis, Lorenz, W.W. (1996) J Biolumin. Chemilumin. 11:31-37; Renilla mulleris, U.S. Pat.No. 6,232,107), b-galactosidase (Nolan, G. et al. (1988) Proc. Natl.Acad. Sci. USA 85:2603-07), b-glucouronidase (Jefferson, R. A. et al.(1987) EMBO J. 6:3901-07; Gallager, S., GUS Protocols: Using the GUSGene as a reporter of gene expression, Academic Press, Inc., 1992), andsecreted form of human placental alkaline phosphatase, SEAP (Cullen, B.R. et al. (1992) Methods Enzymol. 216:362-68). In a preferredembodiment, the codons of the reporter genes are optimized forexpression within a particular organism, especially mammals, andparticularly preferred for human cell expression (see Zolotukhin, S. etal. (1996) J. Virol. 70:4646-54; U.S. Pat. No. 5,968,750; U.S. Pat. No.6,020,192; U.S. Ser. No. 60/290,287, all of which are expresslyincorporate by reference).

[0164] In another embodiment, the reporter protein will bind a labelthat can be used as the basis of the cell enrichment (sorting); that is,the reporter protein serves as an indirect label or detection protein.In this embodiment, the reporter protein is a cell-surface protein. Forexample, the reporter protein may be any cell-surface protein notnormally expressed on the surface of the cell, such that secondarybinding agents serve to distinguish cells that contain the reporterprotein from those that do not. Alternatively, albeit non-preferably,reporters comprising normally expressed cell-surface proteins could beused, and differences between cells containing the reporter constructand those without could be determined. Thus, secondary binding agentsbind to the reporter protein. These secondary binding agents arepreferably labeled, for example with fluors, and can be antibodies,haptens, etc. For example, fluorescently labeled antibodies to thereporter gene can be used as the label. Similarly, membrane-tetheredstreptavidin could serve as a reporter gene, and fluorescently-labeledbiotin could be used as the label, i.e. the secondary binding agent.Alternatively, the secondary binding agents need not be labeled as longas the secondary binding agent can be used to distinguish the cellscontaining the construct; for example, the secondary binding agents maybe used in a column, and the cells passed through, such that theexpression of the reporter gene results in the cell being bound to thecolumn, and a lack of the reporter gene (i.e. inhibition), results inthe cells not being retained on the column. Other suitable reporterproteins/secondary labels include, but are not limited to, antigens andantibodies, enzymes and substrates (or inhibitors), etc.

[0165] In a preferred embodiment, the reporter gene is a survival genethat serves to provide a nucleic acid (or encode a protein) withoutwhich the cell cannot survive, such as drug resistance genes. In thisembodiment, expressing the survival gene allows selection of cellsexpressing the fusion nucleic acid by identifying cells that survive,for example in presence of a selection drug. Examples of drug resistancegenes include, but are not limited to, puromycin resistance(puromycin-N-acetyltransferase) (de la Luna, S. et al. (1992) MethodsEnzymol. 216:376-85), G418 neomycin resistance gene, hygromycinresistance gene (hph), and blasticidine resistance genes (bsr, brs, andBSD; Pere-Gonzalez, et al.(1990) Gene, 86:129-34; Izumi, M. et al.(1991) Exp. Cell Res. 197:229-33; Itaya, M. et al. (1990) J. Biochem.107:799-801; Kimura, M. et al. (1994) Mol. Gen. Genet. 242:121-29). Inaddition, generally applicable survival genes are the family ofATP-binding cassette transporters, including multiple drug resistancegene (MDR1) (see Kane, S. E. et. al. (1988) Mol. Cell. Biol. 8:3316-21and Choi, K. H. et al. (1988) Cell 53:519-29), multi-drug resistanceassociated proteins (MRP) (Bera, T. K. et al. (2001) Mol. Med.7:509-16), and breast cancer associated protein (BCRP or MXR) (Tan, B.et al. (2000) Curr. Opin. Oncol. 12:450-58). When expressed in cells,these selectable genes can confer resistance to a variety of toxicreagents, especially anti-cancer drugs (i.e. methotrexate, colchicine,tamoxifen, mitoxanthrone, and doxorubicin). As will be appreciated bythose skilled in the art, the choice of the selection/survival gene willdepend on the host cell type used.

[0166] In a preferred embodiment, the reporter gene encodes a death genethat causes the cells to die when expressed. Death genes fall into twobasic categories: death genes that encode death proteins requiring adeath ligand to kill the cells, and death genes that encode deathproteins that kill cells as a result of high expression within the celland do not require the addition of any death ligand. Preferred are celldeath mechanisms that requires a two-step process: the expression of thedeath gene and induction of the death phenotype with a signal or ligandsuch that the cells may be grown expressing the death gene, and theninduced to die. A number of death genes/ligand pairs are known,including, but not limited to, the Fas receptor and Fas ligand(Schneider, P. et al. (1997) J. Biol. Chem. 272:18827-33;Gonzalez-Cuadrado, S. et al. (1997) Kidney Int. 51:1739-46; Muruve, D.A. et al. (1997) Hum. Gene Ther. 8:955-63); p450 and cyclophosphamide(Chen, L. et al. (1997) Cancer Res. 57:4830-37); thymidine kinase andgangcylovir (Stone, R. (1992) Science 256:1513), tumor necrosis factor(TNF) receptor and TNF. Alternatively, the death gene need not require aligand, and death results from high expression of the gene; for example,the overexpression of a number of programmed cell death (PCD) proteinsknown to cause cell death, including, but not limited to, caspases, bax,TRADD, FADD, SCK, MEK, etc.

[0167] In a preferred embodiment, death genes also include toxins thatcause cell death, or impair cell survival or cell function whenexpressed by a cell. These toxins generally do not require addition of aligand to produce toxicity. An example of a suitable toxin iscampylobacter toxin CDT (Lara-Tejero, M. (2000) Science, 290:354-57).Expression of CdtB subunit, which has homology to nucleases, causes cellcycle arrest and ultimately cell death. Another toxin, the diphtheriatoxin (and similar Pseudomonas exotoxin), functions by ADP ribosylatingthe ef-2 (elongation factor 2) molecule in the cell and preventingtranslation. Expression of the diphtheria toxin A subunit induces celldeath in cells expressing the toxin fragment. Other useful toxinsinclude cholera toxin and pertussis toxin (catalytic subunit-A ADPribosylates the G protein regulating adenylate cyclase), pierisin fromcabbage butterflys (induces apoptosis in mammalian cells; Watanabe, M.(1999) Proc. Natl. Acad. Sci. USA 96:10608-13), phospholipase snakevenom toxins (Diaz, C. et al. (2001) Arch. Biochem. Biophys. 391:56-64),ribosome inactivating toxins (i.e. ricin A chain, Gluck, A. et al.(1992) J. Mol. Biol. 226:411-24; and nigrin, Munoz, R. et al. (2001)Cancer Lett. 167:163-69) and pore forming toxins (hemolysin andleukocidin). When the target cells are neuronal cells, neuronal specifictoxins may be used to inhibit specific neuronal functions. These includebacterial toxins such as botulinum toxin and tetanus toxin, which areproteases that act on synaptic vesicle associated proteins (i.e.synaptobrevin) to prevent neurotransmitter release (see Binz, T. et al.(1994) J. Biol. Chem. 269:9153-58; Lacy, D. B. et al. (1998) Curr. Opin.Struct. Biol. 8:778-84).

[0168] Another preferred embodiment of a reporter molecule is a cellcycle gene, that is, a gene that causes alterations in the cell cycle.For example, Cdk interacting protein p21 (see Harper, J. W. et al.(1993) Cell 75:805-16), which inhibits cyclin dependent kinases, doesnot cause cell death but causes cell-cycle arrest. Thus, expressing p21allows selecting for regulators of promoter activity or regulators ofp21 activity based on detecting cells that grow out much more quicklydue to low p21 activity, either through inhibiting promoter activity orinactivation of p21 protein activity. As will be appreciated by those inthe art, it is also possible to conFigure the system to select cellsbased on their inability to grow out due to increased p21 activity.Similar mitotic inhibitors include p27, p57, p16, p15, p18 and p19, p19ARF (human homolog p14 ARF). Other cell cycle proteins useful foraltering cell cycle include cyclins (Cln), cyclin dependent kinases(Cdk), cell cycle checkpoint proteins (i.e. Rad17, p53), Cks1 p9, Cdcphosphatases (i.e Cdc 25) etc.

[0169] As is described generally in WO 00/20574, expressly incorporatedby reference, the reporter gene or reporter protein can be part of afusion nucleic acid or fusion polypeptide, and can be attached to acandidate agent at the B or C-terminus, or internally, with or withoutthe use of linkers. In addition, rather than have the reporter gene be afusion partner, it may be located anywhere in the vector being used, orattached to other components.

[0170] In a preferred embodiment, the fusion partner is a targetingsequence. As will be appreciated by those in the art, the localizationof proteins within a cell is a simple method for increasing effectiveconcentration and determining function. For example, RAF1 when localizedto the mitochondrial membrane can inhibit the anti-apoptotic effect ofBCL-2. Similarly, membrane bound Sos induces Ras mediated signaling inT-lymphocytes. These mechanisms are thought to rely on the principle oflimiting the search space for ligands, that is to say, the localizationof a protein to the plasma membrane limits the search for its ligand tothat limited dimensional space near the membrane as opposed to the threedimensional space of the cytoplasm. Alternatively, the concentration ofa protein can also be simply increased by nature of the localization.Shuttling the proteins into the nucleus confines them to a smaller spacethereby increasing concentration. Finally, the ligand or target maysimply be localized to a specific compartment, and inhibitors must belocalized appropriately. Thus, suitable targeting sequences include, butare not limited to, binding sequences capable of causing binding of theexpression product to a predetermined molecule or class of moleculeswhile retaining bioactivity of the expression product, (for example byusing enzyme inhibitor or substrate sequences to target a class ofrelevant enzymes); sequences signaling selective degradation, of itselfor co-bound proteins; and signal sequences capable of constitutivelylocalizing the candidate expression products to a predetermined cellularlocale, including a) subcellular locations such as the golgi,endoplasmic reticulum, nucleus, nucleoli, nuclear membrane,mitochondria, chloroplast, secretory vesicles, lysosome, and cellularmembrane; and b) extracellular locations via a secretory signal.Particularly preferred is localization to either subcellular locationsor to the outside of the cell via secretion.

[0171] In a preferred embodiment, the targeting sequence is a nuclearlocalization signal (NLS). NLSs are generally short, positively charged(basic) domains that serve to direct the entire protein in which theyoccur to the cell's nucleus. Numerous NLS amino acid sequences have beenreported including single basic NLS's such as that of the SV40 (monkeyvirus) large T Antigen (Pro Lys Lys Lys Arg Lys Val), Kalderon (1984),et al., Cell, 39:499-509; the human retinoic acid receptor-β nuclearlocalization signal (ARRRRP); NFkB p50 (EEVQRKRQKL; Ghosh et al., Cell62:1019 (1990); NFkB p65 (EEKRKRTYE; Nolan et al., Cell 64:961 (1991);and others (see for example Boulikas, J. Cell. Biochem. 55(1):32-58(1994), hereby incorporated by reference) and double basic NLS'sexemplified by that of the Xenopus (African clawed toad) protein,nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln AlaLys Lys Lys Lys Leu Asp), Dingwall, et al., Cell, 30:449-458, 1982 andDingwall, et al., J. Cell Biol., 107:641-849; 1988). Numerouslocalization studies have demonstrated that NLSs incorporated insynthetic peptides or grafted onto reporter proteins not normallytargeted to the cell nucleus cause these peptides and reporter proteinsto be concentrated in the nucleus. See, for example, Dingwall, andLaskey, Ann, Rev. Cell Biol., 2:367-390, 1986; Bonnerot, et al., Proc.Natl. Acad. Sci. USA, 84:6795-6799, 1987; Galileo, et al., Proc. Natl.Acad. Sci. USA, 87:458-462, 1990.

[0172] In a preferred embodiment, the targeting sequence is a membraneanchoring signal sequence. This is particularly useful since manyparasites and pathogens bind to the membrane, in addition to the factthat many intracellular events originate at the plasma membrane. Thus,membrane-bound peptide libraries are useful for both the identificationof important elements in these processes as well as for the discovery ofeffective inhibitors. The invention provides methods for presenting therandomized expression product extracellularly or in the cytoplasmicspace; see FIG. 28. For extracellular presentation, a membrane anchoringregion is provided at the carboxyl terminus of the peptide presentationstructure. The randomized expression product region is expressed on thecell surface and presented to the extracellular space, such that it canbind to other surface molecules (affecting their function) or moleculespresent in the extracellular medium. The binding of such molecules couldconfer function on the cells expressing a peptide that binds themolecule. The cytoplasmic region could be neutral or could contain adomain that, when the extracellular randomized expression product regionis bound, confers a function on the cells (activation of a kinase,phosphatase, binding of other cellular components to effect function).Similarly, the randomized expression product-containing region could becontained within a cytoplasmic region, and the transmembrane region andextracellular region remain constant or have a defined function.

[0173] Membrane-anchoring sequences are well known in the art and arebased on the genetic geometry of mammalian transmembrane molecules.Peptides are inserted into the membrane based on a signal sequence(designated herein as ssTM) and require a hydrophobic transmembranedomain (herein TM). The transmembrane proteins are inserted into themembrane such that the regions encoded 5′ of the transmembrane domainare extracellular and the sequences 3′ become intracellular. Of course,if these transmembrane domains are placed 5′ of the variable region,they will serve to anchor it as an intracellular domain, which may bedesirable in some embodiments. ssTMs and TMs are known for a widevariety of membrane bound proteins, and these sequences may be usedaccordingly, either as pairs from a particular protein or with eachcomponent being taken from a different protein, or alternatively, thesequences may be synthetic, and derived entirely from consensus asartificial delivery domains.

[0174] As will be appreciated by those in the art, membrane-anchoringsequences, including both ssTM and TM, are known for a wide variety ofproteins and any of these may be used. Particularly preferredmembrane-anchoring sequences include, but are not limited to, thosederived from CD8, ICAM-2, IL-8R, CD4 and LFA-1.

[0175] Useful sequences include sequences from: 1) class I integralmembrane proteins such as IL-2 receptor beta-chain (residues 1-26 arethe signal sequence, 241-265 are the transmembrane residues; seeHatakeyama et al., Science 244:551 (1989) and von Heijne et al, Eur. J.Biochem. 174:671 (1988)) and insulin receptor beta chain (residues 1-27are the signal, 957-959 are the transmembrane domain and 960-1382 arethe cytoplasmic domain; see Hatakeyama, supra, and Ebina et al., Cell40:747 (1985)); 2) class II integral membrane proteins such as neutralendopeptidase (residues 29-51 are the transmembrane domain, 2-28 are thecytoplasmic domain; see Malfroy et al., Biochem. Biophys. Res. Commun.144:59 (1987)); 3) type III proteins such as human cytochrome P450 NF25(Hatakeyama, supra); and 4) type IV proteins such as humanP-glycoprotein (Hatakeyama, supra). Particularly preferred are CD8 andICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at theextreme 5′ end of the transcript. These consist of the amino acids 1-32in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP; Nakauchi et al.,PNAS USA 82:5126 (1985) and 1-21 in the case of ICAM-2(MSSFGYRTLTVALFTLICCPG; Staunton et al., Nature (London) 339:61 (1989)).These leader sequences deliver the construct to the membrane while thehydrophobic transmembrane domains, placed 3′ of the random candidateregion, serve to anchor the construct in the membrane. Thesetransmembrane domains are encompassed by amino acids 145-195 from CD8(PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR; Nakauchi, supra)and 224-256 from ICAM-2 (MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR; Staunton,supra).

[0176] Alternatively, membrane anchoring sequences include the GPIanchor, which results in a covalent bond between the molecule and thelipid bilayer via a glycosyl-phosphatidylinositol bond for example inDAF (PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT, with the bolded serine thesite of the anchor; see Homans et al., Nature 333(6170):269-72 (1988),and Moran et al., J. Biol. Chem. 266:1250 (1991)). In order to do this,the GPI sequence from Thy-1 can be cassetted 3′ of the variable regionin place of a transmembrane sequence.

[0177] Similarly, myristylation sequences can serve as membraneanchoring sequences. It is known that the myristylation of c-srcrecruits it to the plasma membrane. This is a simple and effectivemethod of membrane localization, given that the first 14 amino acids ofthe protein are solely responsible for this function: MGSSKSKPKDPSQR(see Cross et al., Mol. Cell. Biol. 4(9):1834 (1984); Spencer et al.,Science 262:1019-1024 (1993), both of which are hereby incorporated byreference). This motif has already been shown to be effective in thelocalization of reporter genes and can be used to anchor the zeta chainof the TCR. This motif is placed 5′ of the variable region in order tolocalize the construct to the plasma membrane. Other modifications suchas palmitoylation can be used to anchor constructs in the plasmamembrane; for example, palmitoylation sequences from the Gprotein-coupled receptor kinase GRK6 sequence(LLQRLFSRQDCCGNCSDSEEELPTRL, with the bold cysteines beingpalmitolyated; Stoffel et al., J. Biol. Chem 269:27791 (1994)); fromrhodopsin (KQFRNCMLTSLCCGKNPLGD; Barnstable et al., J. Mol. Neurosci.5(3):207 (1994)); and the p21 H-ras 1 protein (LNPPDESGPGCMSCKCVLS;Capon et al., Nature 302:33 (1983)).

[0178] In a preferred embodiment, the targeting sequence is a lysozomaltargeting sequence, including, for example, a lysosomal degradationsequence such as Lamp-2 (KFERQ; Dice, Ann. N.Y. Acad. Sci. 674:58(1992); or lysosomal membrane sequences from Lamp-1(MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI, Uthayakumar et al., Cell. Mol.Biol. Res. 41:405 (1995)) or Lamp-2(LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF, Konecki et la., Biochem. Biophys.Res. Comm. 205:1-5 (1994), both of which show the transmembrane domainsin italics and the cytoplasmic targeting signal underlined).

[0179] Alternatively, the targeting sequence may be a mitrochondriallocalization sequence, including mitochondrial matrix sequences (e.g.yeast alcohol dehydrogenase Ill; MLRTSSLFTRRVQPSLFSRNILRLQST; Schatz,Eur. J. Biochem. 165:1-6 (1987)); mitochondrial inner membrane sequences(yeast cytochrome c oxidase subunit IV; MLSLRQSIRFFKPATRTLCSSRYLL;Schatz, supra); mitochondrial intermembrane space sequences (yeastcytochrome c1;MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADSLTAEAMTA;Schatz, supra) or mitochondrial outer membrane sequences (yeast 70 kDouter membrane protein; MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKK;Schatz, supra).

[0180] The target sequences may also be endoplasmic reticulum sequences,including the sequences from calreticulin (KDEL; Pelham, Royal SocietyLondon Transactions B; 1-10 (1992)) or adenovirus E3/19K protein(LYLSRRSFIDEKKMP; Jackson et al., EMBO J. 9:3153 (1990).

[0181] Furthermore, targeting sequences also include peroxisomesequences (for example, the peroxisome matrix sequence from Luciferase;SKL; Keller et al., PNAS USA 4:3264 (1987)); farnesylation sequences(for example, P21 H-ras 1; LNPPDESGPGCMSCKCVLS, with the bold cysteinefarnesylated; Capon, supra); geranylgeranylation sequences (for example,protein rab-5A; LTEPTQPTRNQCCSN, with the bold cysteinesgeranylgeranylated; Farnsworth, PNAS USA 91:11963 (1994)); ordestruction sequences (cyclin B1; RTALGDIGN; Klotzbucher et al., EMBO J.1:3053 (1996)).

[0182] In a preferred embodiment, the targeting sequence is a secretorysignal sequence capable of effecting the secretion of the candidatetranslation product. There are a large number of known secretory signalsequences which are placed 5′ to the variable peptide region, and arecleaved from the peptide region to effect secretion into theextracellular space. Secretory signal sequences and theirtransferability to unrelated proteins are well known, e.g., Silhavy, etal. (1985) Microbiol. Rev. 49, 398-418. This is particularly useful togenerate a peptide capable of binding to the surface of, or affectingthe physiology of, a target cell that is other than the host cell, e.g.,the cell infected with the retrovirus. In a preferred approach, a fusionproduct is conFigured to contain, in series, secretion signalpeptide-presentation structure-randomized expression productregion-presentation structure, see FIG. 28. In this manner, target cellsgrown in the vicinity of cells caused to express the library ofpeptides, are bathed in secreted peptide. Target cells exhibiting aphysiological change in response to the presence of a peptide, e.g., bythe peptide binding to a surface receptor or by being internalized andbinding to intracellular targets, and the secreting cells are localizedby any of a variety of selection schemes and the peptide causing theeffect determined. Exemplary effects include variously that of adesigner cytokine (i.e., a stem cell factor capable of causinghematopoietic stem cells to divide and maintain their totipotential), afactor causing cancer cells to undergo spontaneous apoptosis, a factorthat binds to the cell surface of target cells and labels themspecifically, etc.

[0183] Suitable secretory sequences are known, including signals fromIL-2 (MYRMQLLSCIALSLALVTNS; Villinger et al., J. Immunol. 155:3946(1995)), growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT; Roskam et al.,Nucleic Acids Res. 7:30 (1979)); preproinsulin(MALWMRLLPLLALLALWGPDPAAAFVN; Bell et al., Nature 284:26 (1980)); andinfluenza HA protein (MKAKLLVLLYAFVAGDQI; Sekiwawa et al., PNAS80:3563)), with cleavage between the non-underlined-underlined junction.A particularly preferred secretory signal sequence is the signal leadersequence from the secreted cytokine IL4, which comprises the first 24amino acids of IL4 as follows: MGLTSQLLPPLFFLLACAGNFVHG.

[0184] In a preferred embodiment, the fusion partner is a rescuesequence. A rescue sequence is a sequence which may be used to purify orisolate either the candidate agent or the nucleic acid encoding it.Thus, for example, peptide rescue sequences include purificationsequences such as the His₆ tag for use with Ni affinity columns andepitope tags for detection, immunoprecipitation or FACS(fluoroscence-activated cell sorting). Suitable epitope tags include myc(for use with the commercially available 9E10 antibody), the BSPbiotinylation target sequence of the bacterial enzyme BirA, flu tags,lacZ, and GST.

[0185] Alternatively, the rescue sequence may be a unique oligocandidatenucleic acid sequence which serves as a probe target site to allow thequick and easy isolation of the retroviral construct, via PCR, relatedtechniques, or hybridization.

[0186] In a preferred embodiment, the fusion partner is a stabilitysequence to confer stability to the candidate bioactive agent or thenucleic acid encoding it. Thus, for example, peptides may be stabilizedby the incorporation of glycines after the initiation methionine (MG orMGGO), for protection of the peptide to ubiquitination as perVarshavsky=s N-End Rule, thus conferring long half-life in thecytoplasm. Similarly, two prolines at the C-terminus impart peptidesthat are largely resistant to carboxypeptidase action. The presence oftwo glycines prior to the prolines impart both flexibility and preventstructure initiating events in the di-proline to be propagated into thecandidate peptide structure. Thus, preferred stability sequences are asfollows: MG(X)_(n)GGPP, where X is any amino acid and n is an integer ofat least four.

[0187] In one embodiment, the fusion partner is a dimerization sequence.A dimerization sequence allows the non-covalent association of onerandom peptide to another random peptide, with sufficient affinity toremain associated under normal physiological conditions. Thiseffectively allows small libraries of random peptides (for example, 10⁴)to become large libraries if two peptides per cell are generated whichthen dimerize, to form an effective library of 10⁸ (10⁴×10⁴). It alsoallows the formation of longer random peptides, if needed, or morestructurally complex random peptide molecules. The dimers may be homo-or heterodimers.

[0188] Dimerization sequences may be a single sequence thatself-aggregates, or two sequences, each of which is generated in adifferent retroviral construct. That is, nucleic acids encoding both afirst random peptide with dimerization sequence 1, and a second randompeptide with dimerization sequence 2, such that upon introduction into acell and expression of the nucleic acid, dimerization sequence 1associates with dimerization sequence 2 to form a new random peptidestructure.

[0189] Suitable dimerization sequences will encompass a wide variety ofsequences. Any number of protein-protein interaction sites are known. Inaddition, dimerization sequences may also be elucidated using standardmethods such as the yeast two hybrid system, traditional biochemicalaffinity binding studies, or even using the present methods.

[0190] The fusion partners may be placed anywhere (i.e., N-terminal,C-terminal, internal) in the structure as the biology and activitypermits.

[0191] In a preferred embodiment, the fusion partner includes a linkeror tethering sequence. Linker sequences between various targetingsequences (for example, membrane targeting sequences) and the othercomponents of the constructs (such as the randomized candidate agents)may be desirable to allow the candidate agents to interact withpotential targets unhindered. For example, when the candidate bioactiveagent is a peptide, useful linkers include glycine-serine polymers(including, for example, (GS)_(n), (GSGGS)_(n) and (GGGS)_(n), where nis an integer of at least one), glycine-alanine polymers, alanine-serinepolymers, and other flexible linkers such as the tether for the shakerpotassium channel, and a large variety of other flexible linkers, aswill be appreciated by those in the art. Glycine-serine polymers arepreferred since both of these amino acids are relatively unstructured,and therefore may be able to serve as a neutral tether betweencomponents. Secondly, serine is hydrophilic and therefore able tosolubilize what could be a globular glycine chain. Third, similar chainshave been shown to be effective in joining subunits of recombinantproteins such as single chain antibodies.

[0192] In addition, the fusion partners, including presentationstructures, may be modified, randomized, and/or matured to alter thepresentation orientation of the randomized expression product. Forexample, determinants at the base of the loop may be modified toslightly modify the internal loop peptide tertiary structure, whichmaintaining the randomized amino acid sequence.

[0193] In a preferred embodiment, combinations of fusion partners areused. Thus, for example, any number of combinations of presentationstructures, targeting sequences, reporter sequences, rescue sequences,and stability sequences may be used, with or without linker sequences.

[0194] By “candidate nucleic acids” herein is meant nucleic acidscomprising a candidate nucleic acid sequence encoding a candidatebioactive agent. The candidate nucleic acids can be expressed to form acandidate bioactive agent. Therefore, the candidate nucleic acids canfurther encode, for example, a fusion partner and contain sequences toeffect translation or transcription. Where the candidate bioactiveagents are randomized peptides in a peptide library, the candidatenucleic acid generally contains cloning sites which are placed to allowin frame expression of the randomized peptides, and any fusion partners,if present, such as presentation structures. For example, whenpresentation structures are used, the presentation structure willgenerally contain the initiating ATG, as a part of the parent vector.Where the candidate bioactive agents are RNAs in an RNA library, thecandidate nucleic acids are generally constructed with an internal CMVpromoter, tRNA promoter or cell specific promoter designed for immediateand appropriate expression of the RNA structure at the initiation siteof RNA synthesis. The RNA is expressed anti-sense to the direction ofretroviral synthesis and is terminated as known, for example with anorientation specific terminator sequence. Interference from upstreamtranscription is alleviated in the target cell with theself-inactivation deletion, a common feature of certain retroviralexpression systems.

[0195] Generally, the candidate nucleic acids are expressed within thecells to produce expression products of the candidate nucleic acids. Asoutlined above, the expression products include translation products,i.e., peptides, or transcription products, i.e., nucleic acid. Thecandidate bioactive agents and candidate nucleic acids are randomized,either fully randomized or they are biased in their randomization, e.g.in nucleotide/residue frequency generally or per position. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. As is more fully described below, thecandidate nucleic acids which give rise to the candidate expressionproducts are chemically synthesized, and thus may incorporate anynucleotide at any position. Thus, when the candidate nucleic acids areexpressed to form peptides, any amino acid residue may be incorporatedat any position. The synthetic process can be designed to generaterandomized nucleic acids, to allow the formation of all or most of thepossible combinations over the length of the nucleic acid, thus forminga library of randomized candidate nucleic acids.

[0196] The library should provide a sufficiently structurally diversepopulation of randomized expression products to effect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Accordingly, an interactionlibrary must be large enough so that at least one of its members willhave a structure that gives it affinity for some molecule, protein, orother factor whose activity is necessary for completion of the signalingpathway. Although it is difficult to gauge the required absolute size ofan interaction library, nature provides a hint with the immune response:a diversity of 10⁷-10⁸ different antibodies provides at least onecombination with sufficient affinity to interact with most potentialantigens faced by an organism. Published in vitro selection techniqueshave also shown that a library size of 10⁷ to 10⁸ is sufficient to findstructures with affinity for the target. A library of all combinationsof a peptide 7 to 20 amino acids in length, such as proposed here forexpression in retroviruses, has the potential to code for 20⁷ (10⁹) to20²⁰ . Thus, with libraries of 10⁷ to 10⁸ per ml of retroviral particlesthe present methods allow a “working” subset of a theoretically completeinteraction library for 7 amino acids, and a subset of shapes for the20²⁰ library. Thus, in a preferred embodiment, at least 10⁶, preferablyat least 10⁷, more preferably at least 10⁸ and most preferably at least10⁹ different expression products are simultaneously analyzed in thesubject methods. Preferred methods maximize library size and diversity.

[0197] It is important to understand that in any library system encodedby oligonucleotide synthesis one cannot have complete control over thecodons that will eventually be incorporated into the peptide structure.This is especially true in the case of codons encoding stop signals(TAA, TGA, TAG). In a synthesis with NNN as the random region, there isa {fraction (3/64)}, or 4.69%, chance that the codon will be a stopcodon. Thus, in a peptide of 10 residues, there is an unacceptable highlikelihood that 46.7% of the peptides will prematurely terminate. Forfree peptide structures this is perhaps not a problem. But for largerstructures, such as those envisioned here, such termination will lead tosterile peptide expression. To alleviate this, random residues areencoded as NNK, where K=T or G. This allows for encoding of allpotential amino acids (changing their relative representation slightly),but importantly preventing the encoding of two stop residues TAA andTGA. Thus, libraries encoding a 10 amino acid peptide will have a 15.6%chance to terminate prematurely. For candidate nucleic acids which arenot designed to result in peptide expression products, this is notnecessary.

[0198] In one embodiment, the library is fully randomized, with nosequence preferences or constants at any position. In a preferredembodiment, the library is biased. That is, some positions within thesequence are either held constant, or are selected from a limited numberof possibilities. For example, in a preferred embodiment, thenucleotides or amino acid residues are randomized within a definedclass, for example, of hydrophobic amino acids, hydrophilic residues,sterically biased (either small or large) residues, towards the creationof cysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

[0199] In a preferred embodiment, the bias is towards peptides ornucleic acids that interact with known classes of molecules. Forexample, when the candidate bioactive agent is a peptide, it is knownthat much of intracellular signaling is carried out via short regions ofpolypeptides interacting with other polypeptides through small peptidedomains. For instance, a short region from the HIV-1 envelopecytoplasmic domain has been previously shown to block the action ofcellular calmodulin. Regions of the Fas cytoplasmic domain, which showshomology to the mastoparan toxin from Wasps, can be limited to a shortpeptide region with death-inducing apoptotic or G protein inducingfunctions. Magainin, a natural peptide derived from Xenopus, can havepotent anti-tumor and anti-microbial activity. Short peptide fragmentsof a protein kinase C isozyme (βPKC), have been shown to block nucleartranslocation of βPKC in Xenopus oocytes following stimulation. And,short SH-3 target peptides have been used as psuedosubstrates forspecific binding to SH-3 proteins. This is of course a short list ofavailable peptides with biological activity, as the literature is densein this area. Thus, there is much precedent for the potential of smallpeptides to have activity on intracellular signaling cascades. Inaddition, agonists and antagonists of any number of molecules may beused as the basis of biased randomization of candidate bioactive agentsas well.

[0200] Thus, a number of molecules or protein domains are suitable asstarting points for the generation of biased randomized candidatebioactive agents. A large number of small molecule domains are known,that confer a common function, structure or affinity. In addition, as isappreciated in the art, areas of weak amino acid homology may havestrong structural homology. A number of these molecules, domains, and/orcorresponding consensus sequences, are known, including, but are notlimited to, SH-2 domains, SH-3 domains, Pleckstrin, death domains,protease cleavage/recognition sites, enzyme inhibitors, enzymesubstrates, Traf, etc. Similarly, there are a number of known nucleicacid binding proteins containing domains suitable for use in theinvention. For example, leucine zipper consensus sequences are known.

[0201] Where the ultimate expression product is a nucleic acid, at least10, preferably at least 12, more preferably at least 15, most preferablyat least 21 nucleotide positions need to be randomized, with morepreferable if the randomization is less than perfect. Similarly, atleast 5, preferably at least 6 , more preferably at least 7 amino acidpositions need to be randomized; again, more are preferable if therandomization is less than perfect.

[0202] In a preferred embodiment, biased SH-3 domain-bindingoligonucleotides/peptides are made. SH-3 domains have been shown torecognize short target motifs (SH-3 domain-binding peptides), about tento twelve residues in a linear sequence, that can be encoded as shortpeptides with high affinity for the target SH-3 domain. Consensussequences for SH-3 domain binding proteins have been proposed. Thus, ina preferred embodiment, oligos/peptides are made with the followingbiases

[0203] 1. XXXPPXPXX, wherein X is a randomized residue.

[0204] 2. (within the positions of residue positions 11 to −2):

[0205] 11 10 9 8 7 6 5 4 3 2 1

[0206] Met Gly aa11 aa10 aa9 aa8 aa7 Arg Pro Leu Pro Pro hyd

[0207] 0 −1 −2

[0208] Pro hyd hyd Gly Gly Pro Pro STOP

[0209] atg ggc nnk nnk nnk nnk nnk aga cct ctg cct cca sbk ggg sbk sbkgga ggc cca cct TAA1.

[0210] In this embodiment, the N-terminus flanking region is suggestedto have the greatest effects on binding affinity and is thereforeentirely randomized. “Hyd” indicates a bias toward a hydrophobicresidue, i.e.—Val, Ala, Gly, Leu, Pro, Arg. To encode a hydrophobicallybiased residue, “sbk” codon biased structure is used. Examination of thecodons within the genetic code will ensure this encodes generallyhydrophobic residues. s=g,c; b=t, g, c; v=a, g, c; m=a, c; k=t, g; n=a,t, g, c.

[0211] The candidate nucleic acids are introduced into the cells toscreen for bioactive agents capable of altering the phenotype of a cell.By “introduced into” or grammatical equivalents herein is meant that thenucleic acids enter the cells in a manner suitable for subsequentexpression of the nucleic acid. The method of introduction is largelydictated by the targeted cell type, discussed below. Exemplary methodsinclude CaPO₄ precipitation, liposome fusion, lipofectin7,electroporation, viral infection, etc. The candidate nucleic acids maystably integrate into the genome of the host cell (for example, withretroviral introduction, outlined below), or may exist eithertransiently or stably in the cytoplasm (i.e., through the use oftraditional plasmids, utilizing standard regulatory sequences, selectionmarkers, etc.). As many pharmaceutically important screens require humanor model mammalian cell targets, retroviral vectors capable oftransfecting such targets are preferred.

[0212] In a preferred embodiment, the candidate nucleic acids are partof a retroviral particle which infects the cells. Generally, infectionof the cells is straightforward with the application of theinfection-enhancing reagent polybrene, which is a polycation thatfacilitates viral binding to the target cell. Infection can be optimizedsuch that each cell generally expresses a single construct, using theratio of virus particles to number of cells. Infection follows a Poissondistribution.

[0213] In a preferred embodiment, the candidate nucleic acids areintroduced into the cells using retroviral vectors. Currently, the mostefficient nucleic acid transfer methodologies harness the capacity ofengineered viruses, such as retroviruses, to bypass natural cellularbarriers to exogenous nucleic acid uptake. The use of recombinantretroviruses was pioneered by Richard Mulligan and David Baltimore withthe Psi-2 lines and analogous retrovirus packaging systems, based on NIH3T3 cells (see Mann et al., Cell 33:153-159 (1993), hereby incorporatedby reference). Such helper-defective packaging lines are capable ofproducing all the necessary trans proteins -gag, pol, and env- that arerequired for packaging, processing, reverse transcription, andintegration of recombinant genomes. Those RNA molecules that have in cisthe R packaging signal are packaged into maturing virions. Retrovirusesare preferred for a number of reasons. First, their derivation is easy.Second, unlike Adenovirus-mediated nucleic acid delivery, expressionfrom retroviruses is long-term (adenoviruses do not integrate).Adeno-associated viruses have limited space for genes and regulatoryunits and there is some controversy as to their ability to integrate.Retroviruses therefore offer the best current compromise in terms oflong-term expression, genomic flexibility, and stable integration, amongother features. The main advantage of retroviruses is that theirintegration into the host genome allows for their stable transmissionthrough cell division. This ensures that in cell types which undergomultiple independent maturation steps, such as hematopoietic cellprogression, the retrovirus construct will remain resident and continueto express.

[0214] A particularly well suited retroviral transfection system isdescribed in Mann et al., supra: Pear et al., PNAS USA 90(18):8392-6(1993); Kitamura et al., PNAS USA 92:9146-9150 (1995); Kinsella et al.,Human Gene therapy 7:1405-1413; Hofmann et al., PNAS USA 93:5185-5190;Choate et al., Human Gene therapy 7:2247 (1996); and WO 94/19478; andreferences cited therein, all of which are incorporated by reference.

[0215] In one embodiment of the invention, the library is generated in aretrovirus DNA construct backbone, as is generally described in theexamples. Standard oligonucleotide synthesis is done to generate therandom portion of the candidate bioactive agent, using techniques wellknown in the art (see Eckstein, Oligonucleotides and Analogues, APractical Approach, IRL Press at Oxford University Press, 1991);libraries may be commercially purchased. Libraries with up to 10⁹ uniquesequences can be readily generated in such DNA backbones. Aftergeneration of the DNA library, the library is cloned into a firstprimer. The first primer serves as a “cassette” which is inserted intothe retroviral construct. The first primer generally contains a numberof elements, including for example, the required regulatory sequences(e.g. translation, transcription, promoters, eTet), fusion partners,restriction endonuclease (cloning and subcloning) sites, stop codons(preferably in all three frames), regions of complementarity for secondstrand priming (preferably at the end of the stop codon region as minordeletions or insertions may occur in the random region), etc.

[0216] A second primer is then added, which generally consists of someor all of the complementarity region to prime the first primer andoptional necessary sequences for a second unique restriction site forsubcloning. DNA polymerase is added to make double-strandedoligonucleotides. The double-stranded oligonucleotides are cleaved withthe appropriate subcloning restriction endonucleases and subcloned intothe target retroviral vectors, described below.

[0217] Any number of suitable retroviral vectors may be used. Generally,the retroviral vectors may include: selectable marker genes under thecontrol of internal ribosome entry sites (IRES), which allows forbicistronic operons and thus greatly facilitates the selection of cellsexpressing peptides at uniformly high levels; and promoters drivingexpression of a second gene, placed in sense or anti-sense relative tothe 5′ LTR. Suitable selection genes include, but are not limited to,neomycin, blastocidin, bleomycin, puromycin, and hygromycin resistancegenes, as well as self-fluorescent markers such as green fluoroscentprotein, enzymatic markers such as lacZ, and surface proteins such asCD8, etc.

[0218] Preferred vectors include a vector based on the murine stem cellvirus (MSCV) (see Hawley et al., Gene therapy 1:136 (1994)) and amodified MFG virus (Rivere et al., Genetics 92:6733 (1995)), and pBABE,outlined in the examples. A general schematic of the retroviralconstruct is depicted in FIG. 29.

[0219] In a preferred embodiment, a retroviral vector is designed toallow inducible expression of retroviral inserts after integration of asingle vector in target cells. Importantly, the expression system iscontained within the single retrovirus. For example, Tet-inducibleretroviruses have been designed incorporating the Self-Inactivating(SIN) feature of 3′ LTR enhancer/promoter retroviral deletion mutant(Hoffman et al., PNAS USA 93:5185 (1996)). Expression of this vector incells is virtually undetectable in the presence of Tet or analoguesthereof. However, when using the the transactivator tTA, in the absenceof Tet or an analogue thereof, expression is turned on, with uniformincreased expression of the whole population of cells that harbor theinducible retrovirus, indicating that expression is regulated uniformlywithin the infected cell population. Further, using the transactivatorrtTA, in the presence of Tet or an analogue thereof, expression isturned on.

[0220] In this manner the primers create a library of fragments, eachcontaining a different random candidate nucleic acid sequence that mayencode a different peptide (or candidate bioactive agent). The ligationproducts are then transformed into bacteria, such as E. coli, and DNA isprepared from the resulting library, as is generally outlined inKitamura, PNAS USA 92:9146-9150 (1995), hereby expressly incorporated byreference.

[0221] Delivery of the library DNA into a retroviral packaging systemresults in conversion to infectious virus. Suitable retroviral packagingsystem cell lines include, but are not limited to, the Bing and BOSC23cell lines described in WO 94/19478; Soneoka et al., Nucleic Acid Res.23(4):628 (1995); Finer et al., Blood 83:43 (1994); Pheonix packaginglines such as PhiNX-eco and PhiNX-ampho, described below; 292T+gag-poland retrovirus envelope; PA317; and cell lines outlined in Markowitz etal., Virology 167:400 (1988), Markowitz et al., J. Virol. 62:1120(1988), Li et al., PNAS USA 93:11658 (1996), Kinsella et al., Human GeneTherapy 7:1405 (1996), all of which are incorporated by reference.

[0222] Preferred systems include PhiNX-eco and PhiNX-ampho or similarcell lines, which are two cells lines as follows. The cell lines arebased on the BING and BOSC23 cell lines described in WO 94/19478, whichare based on the 293T cell line (a human embryonic kidney linetransformed with adenovirus E1a and carrying a temperature sensitive Tantigen co-selected with neomycin). The unique feature of this cell lineis that it is highly transfectable with either calcium phosphatemediated transfection or lipid-based transfection protocols—greater than50% of 293T cells can be transiently transfected with plasmid DNA. Thus,the cell line could be a cellular milieu in which retroviral structuralproteins and genomic viral RNA could be brought together rapidly forcreation of helper-defective virus. 293T cells were therefore engineeredwith stably integrated defective constructs capable of producinggag-pol, and envelope protein for either ecotropic or amphotropicviruses. These lines are called BOSC23 and Bing, respectively. Theutility of these lines are that one can produce small amounts ofrecombinant virus transiently for use in small-scale experimentation.The lines offer advantages over previously developed stable expressionsystems in that virus can be produced in days rather than months.

[0223] Although the BING and BOSC23 lines are useful in the presentinvention, the PhiNX second-generation lines are preferred. These linesare based on 293T cells as well, and contain the following improvementsover the first-generation lines. First, the ability to monitor gag-polproduction on a cell by cell basis was made by introducing an IRES-CD8surface marker expression cassette downstream of the reading frame ofthe gag-pol construct (other surface markers besides CD8 are alsouseful). IRES (internal ribosome entry site) sequences allow secondaryor tertiary protein translation from a single mRNA transcript. Thus, CD8expression is a direct reflection of intracellular gag-pol and thestability of the producer cell population=s ability to produce gag-polcan be readily monitored by flow cytometry. Second, for both the gag-poland envelope constructs non-Moloney promoters were used to minimizerecombination potential with introduced retroviral constructs, anddifferent promoters for gag-pol and envelope were used to minimize theirinter-recombination potential. The promoters used were CMV and RSV. Twocell lines were created, PHEONIX-ECO and PHEONIX-AMPHO. Gag-pol wasintroduced with hygromycin as the co-selectable marker and the envelopeproteins were introduced with diphtheria resistance as the co-selectablemarker. Finally, the cells were screened to find a relatively rare celltype that produced gag-pol and env in a uniform distribution, althoughthis is not required. In addition, a line termed PHEONIX-gp has beenproduced that expresses only gag-pol. This line is available for furtherpseudotyping of retroviral virions with other envelope proteins such asgibbon ape leukemia virus envelope or Vesicular Stomatitus VSV-Gprotein, Xenotropic, or retargeting envelopes can also be added.

[0224] Both PHEONIX-ECO and PHEONIX-AMPHO were tested for helper virusproduction and established as being helper-virus free. Both lines cancarry episomes for the creation of stable cell lines which can be usedto produce retrovirus. Both lines are readily testable by flow cytometryfor stability of gag-pol (CD8) and envelope expression; after severalmonths of testing the lines appear stable, and do not demonstrate lossof titre as did the first-generation lines BOSC23 and Bing (partly dueto the choice of promoters driving expression of gag-pol and envelope).Both lines can also be used to transiently produce virus in a few days.Thus, these new lines are fully compatible with transient, episomalstable, and library generation for retroviral nucleic acid transferexperiments. Finally, the titres produced by these lines have beentested. Using standard polybrene-enhanced retroviral infection, titresapproaching or above 10⁷ per ml were observed for both PHEONIX-eco andPHEONIX-ampho when carrying episomal constructs. When transientlyproduced virus is made, titres are usually 2 to ⅓ that value.

[0225] These lines are helper-virus free, carry episomes for long-termstable production of retrovirus, stably produce gag-pol and env, and donot demonstrate loss of viral titre over time. In addition, PhiNX-ecoand PhiNX-ampho are capable of producing titres approaching or above 10⁷per ml when carrying episomal constructs, which, with concentration ofvirus, can be enhanced to 10⁸ to 10⁹ per ml.

[0226] In a preferred embodiment, the cell lines disclosed above, andthe other methods for producing retrovirus, are useful for production ofvirus by transient transfection. The virus can either be used directlyor be used to infect another retroviral producer cell line for“expansion” of the library.

[0227] Concentration of virus may be done as follows. Generally,retroviruses are titred by applying retrovirus-containing supernatantonto indicator cells, such as NIH3T3 cells, and then measuring thepercentage of cells expressing phenotypic consequences of infection. Theconcentration of the virus is determined by multiplying the percentageof cells infected by the dilution factor involved, and taking intoaccount the number of target cells available to obtain a relative titre.If the retrovirus contains a reporter gene, such as lacZ, theninfection, integration, and expression of the recombinant virus ismeasured by histological staining for lacZ expression or by flowcytometry (FACS). In general, retroviral titres generated from even thebest of the producer cells do not exceed 10⁷ per ml, unlessconcentration by relatively expensive or exotic apparatus. However, asit has been recently postulated that since a particle as large as aretrovirus will not move very far by brownian motion in liquid, fluiddynamics predicts that much of the virus never comes in contact with thecells to initiate the infection process. However, if cells are grown orplaced on a porous filter and retrovirus is allowed to move past cellsby gradual gravitometric flow, a high concentration of virus aroundcells can be effectively maintained at all times. Thus, up to a ten-foldhigher infectivity by infecting cells on a porous membrane and allowingretrovirus supernatant to flow past them has been seen. This shouldallow titres of 10⁹ after concentration.

[0228] The candidate nucleic acids, as part of the retroviral construct,are introduced into the cells to screen for bioactive agents capable ofaltering the phenotype of a cell, as described herein.

[0229] In a preferred embodiment, a first plurality of cells is screenedusing the methods of the present invention. That is, the cells intowhich the candidate nucleic acids are introduced are screened for analtered phenotype. Thus, in this embodiment, the effect of the bioactiveagent is seen in the same cells in which it is made; i.e., an autocrineeffect.

[0230] In one embodiment, the candidate nucleic acids are introducedinto a first plurality of cells, and the effect of the candidatebioactive agents is screened in a second or third plurality of cells,different from the first plurality of cells, i.e., generally a differentcell type. That is, the effect of the bioactive agents is due to anextracellular effect on a second cell; i.e., an endocrine or paracrineeffect. Using standard techniques, for example, the first plurality ofcells may be grown in or on one media, and the media allowed to touch asecond plurality of cells, and the effect measured. Alternatively, theremay be direct contact between the cells. Thus, “contacting” as usedherein includes both direct and indirect contact and, thus, may be afunctional contact. In this embodiment, the first plurality of cells mayor may not be screened using the methods of the present invention.

[0231] In the methods of the present invention, the cells are treated toconditions suitable for inducing or repressing the expression of thecandidate nucleic acids. The expression products of the candidatenucleic acids may be translation or transcription products.

[0232] Thus, the methods of the present invention comprise introducing amolecular library of randomized candidate nucleic acids into a pluralityof cells, a cellular library. Each of the nucleic acids comprises adifferent, generally randomized, candidate nucleic acid sequence. Theplurality of cells is then screened, as is more fully outlined below,for a cell exhibiting an altered phenotype. The altered cellularphenotype is due to the presence of a candidate bioactive agent.

[0233] By “altered cellular phenotype,” “altered phenotype,” or “changedphysiology” or other grammatical equivalents herein is meant that thephenotype of the cell is altered in a manner that is detectable and/ormeasurable. In a preferred embodiment, the cellular phenotype is altereddue to the presence of a candidate bioactive agent or corresponds to thethe expression of a nucleic acid sequence encoding a candidate bioactiveagent. As will be appreciated in the art, a strength of the presentinvention is the wide variety of cell types and potential phenotypicchanges which may be detected using the present methods of screening.

[0234] Accordingly, any phenotypic change which may be observed,detected, or measured may be the basis of the screening methods herein.Suitable phenotypic changes include, but are not limited to: grossphysical changes such as changes in cell morphology, cell growth, cellviability, adhesion to substrates or other cells, and cellular density;changes in the expression of one or more RNAs, proteins, lipids,hormones, cytokines, or other molecules; changes in the equilibriumstate (i.e., half-life) or one or more RNAs, proteins, lipids, hormones,cytokines, or other molecules; changes in the localization of one ormore RNAs, proteins, lipids, hormones, cytokines, or other molecules;changes in the bioactivity or specific activity of one or more RNAs,proteins, lipids, hormones, cytokines, receptors, or other molecules;changes in the secretion of ions, cytokines, hormones, growth factors,or other molecules; alterations in cellular membrane potentials,polarization, integrity or transport; changes in infectivity,susceptibility, latency, adhesion, and uptake of viruses and bacterialpathogens. By “capable of altering the phenotype” herein is meant thatthe bioactive agent can change the phenotype of the cell in a detectableand/or measurable way.

[0235] The altered phenotype may be detected and selected or sorted orcollected from a parent phenotype, in a wide variety of ways, as isdescribed more fully below, and will generally depend and correspond tothe phenotype that is being changed. Generally, the altered phenotype isdetected using, for example: microscopic analysis of cell morphology;standard cell viability assays, including both increased cell death andincreased cell viability, e.g., cells that are now resistant to celldeath via virus, bacteria, or bacterial or synthetic toxins; standardlabeling assays such as fluorometric indicator assays for the presenceor level of a particular cell or molecule, including FACS or other dyestaining techniques; and other methods of biochemical known in the artfor detection of the expression of the candidate nucleic acid sequence.

[0236] In a preferred embodiment, the candidate nucleic acid sequence isoperably linked to a sequence encoding a reporter protein that is anautofluorescent protein, and the cells having an altered phenotype aresorted or detected or collected by FACs.

[0237] In preferred embodiments, the altered phenotype is detected inthe cell in which the candidate nucleic acid is introduced. In otherembodiments, the altered phenotype is detected in a second cell which isresponding to a molecular signal from a first cell.

[0238] Exemplary assays are described in PCT US97/01019; WO 99/58663; WO00/26241; WO 99/54494; WO 00/72088; and PCT US01/10906, all of which areexpressly incorporated by reference.

[0239] In a preferred embodiment, once a cell with an altered phenotypeis detected using the methods of the invention, the cell can beisolated. This may be performed in any number of ways, as is known inthe art, and will in some instances depend on the assay or screen.Suitable isolation techniques include, but are not limited to, FACS,lysis selection using complement, cell cloning, scanning by Fluorimager,expression of a “survival” protein, induced expression of a cell surfaceprotein or other molecule that can be rendered fluorescent or taggablefor physical isolation; expression of an enzyme that changes anon-fluorescent molecule to a fluorescent one; overgrowth against abackground of no or slow growth; death of cells and isolation of DNA orother cell vitality indicator dyes.

[0240] In a preferred embodiment, the candidate nucleic acid and/or thebioactive agent is isolated from the positive cell. This may beperformed in a number of ways. In a preferred embodiment, primerscomplementary to DNA regions common to the retroviral constructs, or tospecific components of the library such as a rescue sequence, definedabove, are used to “rescue” the unique random sequence. Alternatively,the bioactive agent is isolated using a rescue sequence. Thus, forexample, rescue sequences comprising epitope tags or purificationsequences may be used to pull out the bioactive agent, usingimmunoprecipitation or affinity columns. In some instances, as isoutlined below, this may also pull out the primary target molecule, ifthere is a sufficiently strong binding interaction between the bioactiveagent and the target molecule. Alternatively, the peptide may bedetected using mass spectroscopy.

[0241] Once rescued, the sequence of the bioactive agent and/orbioactive nucleic acid is determined. This information can then be usedin a number of ways.

[0242] In a preferred embodiment, the bioactive agent is resynthesizedand reintroduced into the target cells, to verify the effect. This maybe done using retroviruses, or alternatively using fusions to the HIV-1Tat protein, and analogs and related proteins, which allows very highuptake into target cells. See for example, Fawell et al., PNAS USA91:664 (1994); Frankel et al., Cell 55:1189 (1988); Savion et al., J.Biol. Chem. 256:1149 (1981); Derossi et al., J. Biol. Chem. 269:10444(1994); and Baldin et al., EMBO J. 9:1511 (1990), all of which areincorporated by reference.

[0243] In a preferred embodiment, the sequence of a bioactive agent isused to generate more candidate bioactive agents. For example, thesequence of the bioactive agent may be the basis of a second round of(biased) randomization, to develop bioactive agents with increased oraltered activities. Alternatively, the second round of randomization maychange the affinity of the bioactive agent. Furthermore, it may bedesirable to put the identified random region of the bioactive agentinto other presentation structures, or to alter the sequence of theconstant region of the presentation structure, to alter theconformation/shape of the bioactive agent. It may also be desirable to“walk” around a potential binding site, in a manner similar to themutagenesis of a binding pocket, by keeping one end of the ligand regionconstant and randomizing the other end to shift the binding of thepeptide around.

[0244] In a preferred embodiment, either the bioactive agent or thebioactive nucleic acid encoding it is used to identify target molecules,i.e., the molecules with which the bioactive agent interacts. As will beappreciated by those in the art, there may be primary target molecules,to which the bioactive agent binds or acts upon directly, and there maybe secondary target molecules, which are part of the signalling pathwayaffected by the bioactive agent; these might be termed “validatedtargets”.

[0245] In a preferred embodiment, the bioactive agent is used to pullout target molecules. For example, as outlined herein, if the targetmolecules are proteins, the use of epitope tags or purificationsequences can allow the purification of primary target molecules viabiochemical means (co-immunoprecipitation, affinity columns, etc.).Alternatively, the peptide, when expressed in bacteria and purified, canbe used as a probe against a bacterial cDNA expression library made frommRNA of the target cell type. Or, peptides can be used as “bait” ineither yeast or mammalian two or three hybrid systems. Such interactioncloning approaches have been very useful to isolate DNA-binding proteinsand other interacting protein components. The peptide(s) can be combinedwith other pharmacologic activators to study the epistatic relationshipsof signal transduction pathways in question. It is also possible tosynthetically prepare labeled peptide bioactive agent and use it toscreen a cDNA library expressed in bacteriophage for those cDNAs whichbind the peptide. Furthermore, it is also possible that one could usecDNA cloning via retroviral libraries to “complement” the effect inducedby the peptide. In such a strategy, the peptide would be required to bestochiometrically titrating away some important factor for a specificsignaling pathway. If this molecule or activity is replenished byover-expression of a cDNA from within a cDNA library, then one can clonethe target. Similarly, cDNAs cloned by any of the above yeast orbacteriophage systems can be reintroduced to mammalian cells in thismanner to confirm that they act to complement function in the system thepeptide acts upon.

[0246] Once primary target molecules have been identified, secondarytarget molecules may be identified in the same manner, using the primarytarget as the “bait”. In this manner, signaling pathways may beelucidated. Similarly, bioactive agents specific for secondary targetmolecules may also be discovered, to allow a number of bioactive agentsto act on a single pathway, for example for combination therapies.

[0247] The screening methods of the present invention may be useful toscreen a large number of cell types under a wide variety of conditions.Generally, the host cells are cells that are involved in disease states,and they are tested or screened under conditions that normally result inundesirable consequences on the cells. When a suitable bioactive agentis found, the undesirable effect may be reduced or eliminated andtherefore the cells have an altered phenotype. Alternatively, with aneye towards elucidating the cellular mechanisms associated with thedisease state or signaling pathway, normally desirable consequences maybe reduced or eliminated and therefore the cells have an alteredphenotype.

[0248] In a preferred embodiment, the present methods are useful incancer applications. The ability to rapidly and specifically kill tumorcells is a cornerstone of cancer chemotherapy. In general, using themethods of the present invention, random libraries can be introducedinto any tumor cell (primary or cultured), and peptides identified whichby themselves induce apoptosis, cell death, loss of cell division ordecreased cell growth. This may be done de novo, or by biasedrandomization toward known peptide agents, such as angiostatin, whichinhibits blood vessel wall growth. Alternatively, the methods of thepresent invention can be combined with other cancer therapeutics (e.g.drugs or radiation) to sensitize the cells and thus induce rapid andspecific apoptosis, cell death, loss of cell division or decreased cellgrowth after exposure to a secondary agent. Similarly, the presentmethods may be used in conjunction with known cancer therapeutics toscreen for agonists to make the therapeutic more effective or lesstoxic. This is particularly preferred when the chemotherapeutic is veryexpensive to produce such as taxol.

[0249] Known oncogenes such as v-Abl, v-Src, v-Ras, and others, induce atransformed phenotype leading to abnormal cell growth when transfectedinto certain cells. This is also a major problem with micrometastases.Thus, in a preferred embodiment, non-transformed cells can betransfected with these oncogenes, and then random libraries introducedinto these cells, to select for bioactive agents which reverse orcorrect the transformed state. One of the signal features of onconucleicacid transformation of cells is the loss of contact inhibition and theability to grow in soft-agar. When transforming viruses are constructedcontaining v-Abl, v-Src, or v-Ras in IRES-puro retroviral vectors,infected into target 3T3 cells, and subjected to puromycin selection,all of the 3T3 cells hyper-transform and detach from the plate. Thecells may be removed by washing with fresh medium. This can serve as thebasis of a screen, since cells which express a bioactive agent willremain attached to the plate and form colonies.

[0250] Similarly, the growth and/or spread of certain tumor types isenhanced by stimulatory responses from growth factors and cytokines(PDGF, EGF, Heregulin, and others) which bind to receptors on thesurfaces of specific tumors. In a preferred embodiment, the methods ofthe invention are used to inhibit or stop tumor growth and/or spread, byfinding bioactive agents capable of blocking the ability of the growthfactor or cytokine to stimulate the tumor cell. The introduction ofrandom libraries into specific tumor cells with the addition of thegrowth factor or cytokine, followed by selection of bioactive agentswhich block the binding, signaling, phenotypic and/or functionalresponses of these tumor cells to the growth factor or cytokine inquestion.

[0251] Similarly, the spread of cancer cells (invasion and metastasis)is a significant problem limiting the success of cancer therapies. Theability to inhibit the invasion and/or migration of specific tumor cellswould be a significant advance in the therapy of cancer. Tumor cellsknown to have a high metastatic potential (for example, melanoma, lungcell carcinoma, breast and ovarian carcinoma) can have random librariesintroduced into them, and peptides selected which in a migration orinvasion assay, inhibit the migration and/or invasion of specific tumorcells. Particular applications for inhibition of the metastaticphenotype, which could allow a more specific inhibition of metastasis,include the metastasis suppressor gene NM23, which codes for adinucleoside diphosphate kinase. Thus intracellular peptide activatorsof this gene could block metastasis, and a screen for its upregulation(by fusing it to a reporter gene) would be of interest. Many oncogenesalso enhance metastasis. Peptides which inactivate or counteract mutatedRAS oncogenes, v-MOS, v-RAF, A-RAF, v-SRC, v-FES, and v-FMS would alsoact as anti-metastatics. Peptides which act intracellularly to block therelease of combinations of proteases required for invasion, such as thematrix metalloproteases and urokinase, could also be effectiveantimetastatics.

[0252] In a preferred embodiment, the random libraries of the presentinvention are introduced into tumor cells known to have inactivatedtumor suppressor genes, and successful reversal by either reactivationor compensation of the knockout would be screened by restoration of thenormal phenotype. A major example is the reversal of p53-inactivatingmutations, which are present in 50% or more of all cancers. Since p53'sactions are complex and involve its action as a transcription factor,there are probably numerous potential ways a peptide or small moleculederived from a peptide could reverse the mutation. One example would beupregulation of the immediately downstream cyclin-dependent kinasep21CIP1/WAF1. To be useful such reversal would have to work for many ofthe different known p53 mutations. This is currently being approached bygene therapy; one or more small molecules which do this might bepreferable.

[0253] Another example involves screening of bioactive agents whichrestore the constitutive function of the brca-1 or brca-2 genes, andother tumor suppressor genes important in breast cancer such as theadenomatous polyposis coli nucleic acid (APC) and the Drosophiladiscs-large nucleic acid (Dig), which are components of cell-celljunctions. Mutations of brca-1 are important in hereditary ovarian andbreast cancers, and constitute an additional application of the presentinvention.

[0254] In a preferred embodiment, the methods of the present inventionare used to create novel cell lines from cancers from patients. Aretrovirally delivered short peptide which inhibits the final commonpathway of programmed cell death should allow for short- and possiblylong-term cell lines to be established. Conditions of in vitro cultureand infection of human leukemia cells will be established. There is areal need for methods which allow the maintenance of certain tumor cellsin culture long enough to allow for physiological and pharmacologicalstudies. Currently, some human cell lines have been established by theuse of transforming agents such as Epstein-Barr virus that considerablyalters the existing physiology of the cell. On occasion, cells will growon their own in culture but this is a random event. Programmed celldeath (apoptosis) occurs via complex signaling pathways within cellsthat ultimately activate a final common pathway producing characteristicchanges in the cell leading to a non-inflammatory destruction of thecell. It is well known that tumor cells have a high apoptotic index, orpropensity to enter apoptosis in vivo. When cells are placed in culture,the in vivo stimuli for malignant cell growth are removed and cellsreadily undergo apoptosis. The objective would be to develop thetechnology to establish cell lines from any number of primary tumorcells, for example primary human leukemia cells, in a reproduciblemanner without altering the native conFiguration of the signalingpathways in these cells. By introducing nucleic acids encoding peptideswhich inhibit apoptosis, increased cell survival in vitro, and hence theopportunity to study signaling transduction pathways in primary humantumor cells, is accomplished. In addition, these methods may be used forculturing primary cells, i.e., non-tumor cells.

[0255] In a preferred embodiment, the present methods are useful incardiovascular applications. In a preferred embodiment, cardiomyocytesmay be screened for the prevention of cell damage or death in thepresence of normally injurious conditions, including, but not limitedto, the presence of toxic drugs (particularly chemotherapeutic drugs),for example, to prevent heart failure following treatment withadriamycin; anoxia, for example in the setting of coronary arteryocclusion; and autoimmune cellular damage by attack from activatedlymphoid cells (for example as seen in post viral myocarditis andlupus). Candidate bioactive agents are inserted into cardiomyocytes, thecells are subjected to the insult, and bioactive agents are selectedthat prevent any or all of: apoptosis; membrane depolarization (i.e.,decrease arrythmogenic potential of insult); cell swelling; or leakageof specific intracellular ions, second messengers and activatingmolecules (for example, arachidonic acid and/or lysophosphatidic acid).

[0256] In a preferred embodiment, the present methods are used to screenfor diminished arrhythmia potential in cardiomyocytes. The screenscomprise the introduction of the candidate nucleic acids encodingcandidate bioactive agents, followed by the application of arrythmogenicinsults, with screening for bioactive agents that block specificdepolarization of cell membrane. This may be detected using patchclamps, or via fluorescence techniques). Similarly, channel activity(for example, potassium and chloride channels) in cardiomyocytes couldbe regulated using the present methods in order to enhance contractilityand prevent or diminish arrhythmias.

[0257] In a preferred embodiment, the present methods are used to screenfor enhanced contractile properties of cardiomyocytes and diminish heartfailure potential. The introduction of the libraries of the inventionfollowed by measuring the rate of change of myosinpolymerization/depolymerization using fluorescent techniques can bedone. Bioactive agents which increase the rate of change of thisphenomenon can result in a greater contractile response of the entiremyocardium, similar to the effect seen with digitalis.

[0258] In a preferred embodiment, the present methods are useful toidentify agents that will regulate the intracellular and sarcolemmalcalcium cycling in cardiomyocytes in order to prevent arrhythmias.Bioactive agents are selected that regulate sodium-calcium exchange,sodium proton pump function, and regulation of calcium-ATPase activity.

[0259] In a preferred embodiment, the present methods are useful toidentify agents that diminish embolic phenomena in arteries andarterioles leading to strokes (and other occlusive events leading tokidney failure and limb ischemia) and angina precipitating a myocardialinfarct are selected. For example, bioactive agents which will diminishthe adhesion of platelets and leukocytes, and thus diminish theocclusion events. Adhesion in this setting can be inhibited by thelibraries of the invention being inserted into endothelial cells(quiescent cells, or activated by cytokines, i.e., IL-1, and growthfactors, i.e., PDGF/EGF) and then screening for peptides that either: 1)downregulate adhesion molecule expression on the surface of theendothelial cells (binding assay); 2) block adhesion molecule activationon the surface of these cells (signaling assay); or 3) release in anautocrine manner peptides that block receptor binding to the cognatereceptor on the adhering cell.

[0260] Embolic phenomena can also be addressed by activating proteolyticenzymes on the cell surfaces of endothelial cells, and thus releasingactive enzyme which can digest blood clots. Thus, delivery of thelibraries of the invention to endothelial cells is done, followed bystandard fluorogenic assays, which will allow monitoring of proteolyticactivity on the cell surface towards a known substrate. Bioactive agentscan then be selected which activate specific enzymes towards specificsubstrates.

[0261] In a preferred embodiment, arterial inflammation in the settingof vasculitis and post-infarction can be regulated by decreasing thechemotactic responses of leukocytes and mononuclear leukocytes. This canbe accomplished by blocking chemotactic receptors and their respondingpathways on these cells. Candidate bioactive libraries can be insertedinto these cells, and the chemotactic response to diverse chemokines(for example, to the IL-8 family of chemokines, RANTES) inhibited incell migration assays.

[0262] In a preferred embodiment, arterial restenosis following coronaryangioplasty can be controlled by regulating the proliferation ofvascular intimal cells and capillary and/or arterial endothelial cells.Candidate bioactive agent libraries can be inserted into these celltypes and their proliferation in response to specific stimuli monitored.One application may be intracellular peptides which block the expressionor function of c-myc and other oncogenes in smooth muscle cells to stoptheir proliferation. A second application may involve the expression oflibraries in vascular smooth muscle cells to selectively induce theirapoptosis. Application of small molecules derived from these peptidesmay require targeted drug delivery; this is available with stents,hydrogel coatings, and infusion-based catheter systems. Peptides whichdownregulate endothelin-1A receptors or which block the release of thepotent vasoconstrictor and vascular smooth muscle cell mitogenendothelin-1 may also be candidates for therapeutics. Peptides can beisolated from these libraries which inhibit growth of these cells, orwhich prevent the adhesion of other cells in the circulation known torelease autocrine growth factors, such as platelets (PDGF) andmononuclear leukocytes.

[0263] The control of capillary and blood vessel growth is an importantgoal in order to promote increased blood flow to ischemic areas(growth), or to cut-off the blood supply (angiogenesis inhibition) oftumors. Candidate bioactive agent libraries can be inserted intocapillary endothelial cells and their growth monitored. Stimuli such aslow oxygen tension and varying degrees of angiogenic factors canregulate the responses, and peptides isolated that produce theappropriate phenotype. Screening for antagonism of vascular endothelialcell growth factor, important in angiogenesis, would also be useful.

[0264] In a preferred embodiment, the present methods are useful inscreening for decreases in atherosclerosis producing mechanisms to findpeptides that regulate LDL and HDL metabolism. Candidate libraries canbe inserted into the appropriate cells (including hepatocytes,mononuclear leukocytes, endothelial cells) and peptides selected whichlead to a decreased release of LDL or diminished synthesis of LDL, orconversely to an increased release of HDL or enhanced synthesis of HDL.Bioactive agents can also be isolated from candidate libraries whichdecrease the production of oxidized LDL, which has been implicated inatherosclerosis and isolated from atherosclerotic lesions. This couldoccur by decreasing its expression, activating reducing systems orenzymes, or blocking the activity or production of enzymes implicated inproduction of oxidized LDL, such as 15-lipoxygenase in macrophages.

[0265] In a preferred embodiment, the present methods are used inscreens to regulate obesity via the control of food intake mechanisms ordiminishing the responses of receptor signaling pathways that regulatemetabolism. Bioactive agents that regulate or inhibit the responses ofneuropeptide Y (NPY), cholecystokinin and galanin receptors, areparticularly desirable. Candidate libraries can be inserted into cellsthat have these receptors cloned into them, and inhibitory peptidesselected that are secreted in an autocrine manner that block thesignaling responses to galanin and NPY. In a similar manner, peptidescan be found that regulate the leptin receptor.

[0266] In a preferred embodiment, the present methods are useful inneurobiology applications. Candidate libraries may be used for screeningfor anti-apoptotics for preservation of neuronal function and preventionof neuronal death. Initial screens would be done in cell culture. Oneapplication would include prevention of neuronal death, by apoptosis, incerebral ischemia resulting from stroke. Apoptosis is known to beblocked by neuronal apoptosis inhibitory protein (NAIP); screens for itsupregulation, or effecting any coupled step could yield peptides whichselectively block neuronal apoptosis. Other applications includeneurodegenerative diseases such as Alzheimer's disease and Huntington'sdisease.

[0267] In a preferred embodiment, the present methods are useful in bonebiology applications. Osteoclasts are known to play a key role in boneremodeling by breaking down “old” bone, so that osteoblasts can lay down“new” bone. In osteoporosis one has an imbalance of this process.Osteoclast overactivity can be regulated by inserting candidatelibraries into these cells, and then looking for bioactive agents thatproduce: 1) a diminished processing of collagen by these cells; 2)decreased pit formation on bone chips; and 3) decreased release ofcalcium from bone fragments.

[0268] The present methods may also be used to screen for agonists ofbone morphogenic proteins, hormone mimetics to stimulate, regulate, orenhance new bone formation (in a manner similar to parathyroid hormoneand calcitonin, for example). These have use in osteoporosis, for poorlyhealing fractures, and to accelerate the rate of healing of newfractures. Furthermore, cell lines of connective tissue origin can betreated with candidate libraries and screened for their growth,proliferation, collagen stimulating activity, and/or prolineincorporating ability on the target osteoblasts. Alternatively,candidate libraries can be expressed directly in osteoblasts orchondrocytes and screened for increased production of collagen or bone.

[0269] In a preferred embodiment, the present methods are useful in skinbiology applications. Keratinocyte responses to a variety of stimuli mayresult in psoriasis, a proliferative change in these cells. Candidatelibraries can be inserted into cells removed from active psoriaticplaques, and bioactive agents isolated which decrease the rate of growthof these cells.

[0270] In a preferred embodiment, the present methods are useful in theregulation or inhibition of keloid formation (i.e., excessive scarring).Candidate libraries inserted into skin connective tissue cells isolatedfrom individuals with this condition, and bioactive agents isolated thatdecrease proliferation, collagen formation, or proline incorporation.Results from this work can be extended to treat the excessive scarringthat also occurs in burn patients. If a common peptide motif is found inthe context of the keloid work, then it can be used widely in a topicalmanner to diminish scarring post burn.

[0271] Similarly, wound healing for diabetic ulcers and other chronic“failure to heal” conditions in the skin and extremities can beregulated by providing additional growth signals to cells which populatethe skin and dermal layers. Growth factor mimetics may in fact be veryuseful for this condition. Candidate libraries can be inserted into skinconnective tissue cells, and bioactive agents isolated which promote thegrowth of these cells under “harsh” conditions, such as low oxygentension, low pH, and the presence of inflammatory mediators.

[0272] Cosmeceutical applications of the present invention include thecontrol of melanin production in skin melanocytes. A naturally occurringpeptide, arbutin, is a tyrosine hydroxylase inhibitor, a key enzyme inthe synthesis of melanin. Candidate libraries can be inserted intomelanocytes and known stimuli that increase the synthesis of melaninapplied to the cells. Bioactive agents can be isolated that inhibit thesynthesis of melanin under these conditions.

[0273] In a preferred embodiment, the present methods are useful inendocrinology applications. The retroviral peptide library technologycan be applied broadly to any endocrine, growth factor, cytokine orchemokine network which involves a signaling peptide or protein thatacts in either an endocrine, paracrine or autocrine manner that binds ordimerizes a receptor and activates a signaling cascade that results in aknown phenotypic or functional outcome. The methods are applied so as toisolate a peptide which either mimics the desired hormone (i.e.,insulin, leptin, calcitonin, PDGF, EGF, EPO, GMCSF, IL1-17, mimetics) orinhibits its action by either blocking the release of the hormone,blocking its binding to a specific receptor or carrier protein (forexample, CRF binding protein), or inhibiting the intracellular responsesof the specific target cells to that hormone. Selection of peptideswhich increase the expression or release of hormones from the cellswhich normally produce them could have broad applications to conditionsof hormonal deficiency.

[0274] In a preferred embodiment, the present methods are useful ininfectious disease applications. Viral latency (herpes viruses such asCMV, EBV, HBV, and other viruses such as HIV) and their reactivation area significant problem, particularly in immunosuppressed patients (patients with AIDS and transplant patients). The ability to block thereactivation and spread of these viruses is an important goal. Celllines known to harbor or be susceptible to latent viral infection can beinfected with the specific virus, and then stimuli applied to thesecells which have been shown to lead to reactivation and viralreplication. This can be followed by measuring viral titers in themedium and scoring cells for phenotypic changes. Candidate libraries canthen be inserted into these cells under the above conditions, andpeptides isolated which block or diminish the growth and/or release ofthe virus. As with chemotherapeutics, these experiments can also be donewith drugs which are only partially effective towards this outcome, andbioactive agents isolated which enhance the virucidal effect of thesedrugs.

[0275] One example of many is the ability to block HIV-1 infection.HIV-1 requires CD4 and a co-receptor which can be one of several seventransmembrane G-protein coupled receptors. In the case of the infectionof macrophages, CCR-5 is the required co-receptor, and there is strongevidence that a block on CCR-5 will result in resistance to HIV-1infection. There are two lines of evidence for this statement. First, itis known that the natural ligands for CCR-5, the CC chemokines RANTES,MIPla and MIP1b are responsible for CD8+ mediated resistance to HIV.Second, individuals homozygous for a mutant allele of CCR-5 arecompletely resistant to HIV infection. Thus, an inhibitor of theCCR5/HIV interaction would be of enormous interest to both biologistsand clinicians. The extracellular anchored constructs offer superb toolsfor such a discovery. Into the transmembrane, epitope tagged,glycine-serine tethered constructs (ssTM V G20 E TM), one can place arandom, cyclized peptide library of the general sequence CNNNNNNNNNNC orC-(X)_(n)-C. Then one infects a cell line that expresses CCR-5 withretroviruses containing this library. Using an antibody to CCR-5 one canuse FACS to sort desired cells based on the binding of this antibody tothe receptor. All cells which do not bind the antibody will be assumedcontain inhibitors of this antibody binding site. These inhibitors, inthe retroviral construct can be further assayed for their ability toinhibit HIV-1 entry. Viruses are known to enter cells using specificreceptors to bind to cells (for example, HIV uses CD4, coronavirus usesCD13, murine leukemia virus uses transport protein, and measles virususes CD44) and to fuse with cells (HIV uses chemokine receptor).Candidate libraries can be inserted into target cells known to bepermissive to these viruses, and bioactive agents isolated which blockthe ability of these viruses to bind and fuse with specific targetcells.

[0276] In a preferred embodiment, the present invention finds use withinfectious organisms. Intracellular organisms such as mycobacteria,listeria, salmonella, pneumocystis, yersinia, leishmania, T. cruzi, canpersist and replicate within cells, and become active inimmunosuppressed patients. There are currently drugs on the market andin development which are either only partially effective or ineffectiveagainst these organisms. Candidate libraries can be inserted intospecific cells infected with these organisms (pre- or post-infection),and bioactive agents selected which promote the intracellulardestruction of these organisms in a manner analogous to intracellular“antibiotic peptides” similar to magainins. In addition peptides can beselected which enhance the cidal properties of drugs already underinvestigation which have insufficient potency by themselves, but whencombined with a specific peptide from a candidate library, aredramatically more potent through a synergistic mechanism. Finally,bioactive agents can be isolated which alter the metabolism of theseintracellular organisms, in such a way as to terminate theirintracellular life cycle by inhibiting a key organismal event.

[0277] Antibiotic drugs that are widely used have certain dosedependent, tissue specific toxicities. For example renal toxicity isseen with the use of gentamicin, tobramycin, and amphotericin;hepatotoxicity is seen with the use of INH and rifampin; bone marrowtoxicity is seen with chloramphenicol; and platelet toxicity is seenwith ticarcillin, etc. These toxicities limit their use. Candidatelibraries can be introduced into the specific cell types where specificchanges leading to cellular damage or apoptosis by the antibiotics areproduced, and bioactive agents can be isolated that confer protection,when these cells are treated with these specific antibiotics.

[0278] Furthermore, the present invention finds use in screening forbioactive agents that block antibiotic transport mechanisms. The rapidsecretion from the blood stream of certain antibiotics limits theirusefulness. For example penicillins are rapidly secreted by certaintransport mechanisms in the kidney and choroid plexus in the brain.Probenecid is known to block this transport and increase serum andtissue levels. Candidate agents can be inserted into specific cellsderived from kidney cells and cells of the choroid plexus known to haveactive transport mechanisms for antibiotics. Bioactive agents can thenbe isolated which block the active transport of specific antibiotics andthus extend the serum halflife of these drugs.

[0279] In a preferred embodiment, the present methods are useful in drugtoxicities and drug resistance applications. Drug toxicity is asignificant clinical problem. This may manifest itself as specifictissue or cell damage with the result that the drug=s effectiveness islimited. Examples include myeloablation in high dose cancerchemotherapy, damage to epithelial cells lining the airway and gut, andhair loss. Specific examples include adriamycin induced cardiomyocytedeath, cisplatinin-induced kidney toxicity, vincristine-induced gutmotility disorders, and cyclosporin-induced kidney damage. Candidatelibraries can be introduced into specific cell types with characteristicdrug-induced phenotypic or functional responses, in the presence of thedrugs, and agents isolated which reverse or protect the specific celltype against the toxic changes when exposed to the drug. These effectsmay manifest as blocking the drug induced apoptosis of the cell ofinterest, thus initial screens will be for survival of the cells in thepresence of high levels of drugs or combinations of drugs used incombination chemotherapy.

[0280] Drug toxicity may be due to a specific metabolite produced in theliver or kidney which is highly toxic to specific cells, or due to druginteractions in the liver which block or enhance the metabolism of anadministered drug. Candidate libraries can be introduced into liver orkidney cells following the exposure of these cells to the drug known toproduce the toxic metabolite. Bioactive agents can be isolated whichalter how the liver or kidney cells metabolize the drug, and specificagents identified which prevent the generation of a specific toxicmetabolite. The generation of the metabolite can be followed by massspectrometry, and phenotypic changes can be assessed by microscopy. Sucha screen can also be done in cultured hepatocytes, cocultured withreadout cells which are specifically sensitive to the toxic metabolite.Applications include reversible (to limit toxicity) inhibitors ofenzymes involved in drug metabolism.

[0281] Multiple drug resistance, and hence tumor cell selection,outgrowth, and relapse, leads to morbidity and mortality in cancerpatients. Candidate libraries can be introduced into tumor cell lines(primary and cultured) that have demonstrated specific or multiple drugresistance. Bioactive agents can then be identified which confer drugsensitivity when the cells are exposed to the drug of interest, or todrugs used in combination chemotherapy. The readout can be the onset ofapoptosis in these cells, membrane permeability changes, the release ofintracellular ions and fluorescent markers. The cells in which multidrugresistance involves membrane transporters can be preloaded withfluorescent transporter substrates, and selection carried out forpeptides which block the normal efflux of fluorescent drug from thesecells. Candidate libraries are particularly suited to screening forpeptides which reverse poorly characterized or recently discoveredintracellular mechanisms of resistance or mechanisms for which few or nochemosensitizers currently exist, such as mechanisms involving LRP (lungresistance protein). This protein has been implicated in multidrugresistance in ovarian carcinoma, metastatic malignant melanoma, andacute myeloid leukemia. Particularly interesting examples includescreening for agents which reverse more than one important resistancemechanism in a single cell, which occurs in a subset of the most drugresistant cells, which are also important targets. Applications wouldinclude screening for peptide inhibitors of both MRP (multidrugresistance related protein) and LRP for treatment of resistant cells inmetastatic melanoma, for inhibitors of both p-glycoprotein and LRP inacute myeloid leukemia, and for inhibition (by any mechanism) of allthree proteins for treating pan-resistant cells.

[0282] In a preferred embodiment, the present methods are useful inimproving the performance of existing or developmental drugs. First passmetabolism of orally administered drugs limits their oralbioavailability, and can result in diminished efficacy as well as theneed to administer more drug for a desired effect. Reversible inhibitorsof enzymes involved in first pass metabolism may thus be a usefuladjunct enhancing the efficacy of these drugs. First pass metabolismoccurs in the liver, thus inhibitors of the corresponding catabolicenzymes may enhance the effect of the cognate drugs. Reversibleinhibitors would be delivered at the same time as, or slightly before,the drug of interest. Screening of candidate libraries in hepatocytesfor inhibitors (by any mechanism, such as protein downregulation as wellas a direct inhibition of activity) of particularly problematicalisozymes would be of interest. These include the CYP3A4 isozymes ofcytochrome P450, which are involved in the first pass metabolism of theanti-HIV drugs saquinavir and indinavir. Other applications couldinclude reversible inhibitors of UDP-glucuronyltransferases,sulfotransferases, N-acetyltransferases, epoxide hydrolases, andglutathione S-transferases, depending on the drug. Screens would be donein cultured hepatocytes or liver microsomes, and could involveantibodies recognizing the specific modification performed in the liver,or cocultured readout cells, if the metabolite had a differentbioactivity than the untransformed drug. The enzymes modifying the drugwould not necessarily have to be known, if screening was for lack ofalteration of the drug.

[0283] In a preferred embodiment, the present methods are useful inimmunobiology, inflammation, and allergic response applications.Selective regulation of T lymphocyte responses is a desired goal inorder to modulate immune-mediated diseases in a specific manner.Candidate libraries can be introduced into specific T cell subsets (TH1,TH2, CD4+, CD8+, and others) and the responses which characterize thosesubsets (cytokine generation, cytotoxicity, proliferation in response toantigen being presented by a mononuclear leukocyte, and others) modifiedby members of the library. Agents can be selected which increase ordiminish the known T cell subset physiologic response. This approachwill be useful in any number of conditions, including: 1) autoimmunediseases where one wants to induce a tolerant state (select a peptidethat inhibits T cell subset from recognizing a self-antigen bearingcell); 2) allergic diseases where one wants to decrease the stimulationof IgE producing cells (select peptide which blocks release from T cellsubsets of specific B-cell stimulating cytokines which induce switch toIgE production); 3) in transplant patients where one wants to induceselective immunosuppression (select peptide that diminishesproliferative responses of host T cells to foreign antigens); 4) inlymphoproliferative states where one wants to inhibit the growth orsensitize a specific T cell tumor to chemotherapy and/or radiation; 5)in tumor surveillance where one wants to inhibit the killing ofcytotoxic T cells by Fas ligand bearing tumor cells; and 5) in T cellmediated inflammatory diseases such as Rheumatoid arthritis, Connectivetissue diseases (SLE), Multiple sclerosis, and inflammatory boweldisease, where one wants to inhibit the proliferation of disease-causingT cells (promote their selective apoptosis) and the resulting selectivedestruction of target tissues (cartilage, connective tissue,oligodendrocytes, gut endothelial cells, respectively).

[0284] Regulation of B cell responses will permit a more selectivemodulation of the type and amount of immunoglobulin made and secreted byspecific B cell subsets. Candidate libraries can be inserted into Bcells and bioactive agents selected which inhibit the release andsynthesis of a specific immunoglobulin. This may be useful in autoimmunediseases characterized by the overproduction of auto antibodies and theproduction of allergy causing antibodies, such as IgE. Agents can alsobe identified which inhibit or enhance the binding of a specificimmunoglobulin subclass to a specific antigen either foreign of self.Finally, agents can be selected which inhibit the binding of a specificimmunoglobulin subclass to its receptor on specific cell types.

[0285] Similarly, agents which affect cytokine production may beselected, generally using two cell systems. For example, cytokineproduction from macrophages, monocytes, etc. may be evaluated.Similarly, agents which mimic cytokines, for example erythropoetin andIL1-17, may be selected, or agents that bind cytokines such as TNF-″,before they bind their receptor.

[0286] Antigen processing by mononuclear leukocytes (ML) is an importantearly step in the immune system=s ability to recognize and eliminateforeign proteins. Candidate agents can be inserted into ML cell linesand agents selected which alter the intracellular processing of foreignpeptides and sequence of the foreign peptide that is presented to Tcells by MLs on their cell surface in the context of Class II MHC. Onecan look for members of the library that enhance immune responses of aparticular T cell subset (for example, the peptide would in fact work asa vaccine), or look for a library member that binds more tightly to MHC,thus displacing naturally occurring peptides, but nonetheless the agentwould be less immunogenic (less stimulatory to a specific T cell clone).This agent would in fact induce immune tolerance and/or diminish immuneresponses to foreign proteins. This approach could be used intransplantation, autoimmune diseases, and allergic diseases.

[0287] The release of inflammatory mediators (cytokines, leukotrienes,prostaglandins, platelet activating factor, histamine, neuropeptides,and other peptide and lipid mediators) is a key element in maintainingand amplifying aberrant immune responses. Candidate libraries can beinserted into MLs, mast cells, eosinophils, and other cellsparticipating in a specific inflammatory response, and bioactive agentsselected which inhibit the synthesis, release and binding to the cognatereceptor of each of these types of mediators.

[0288] In a preferred embodiment, the present methods are useful inbiotechnology applications. Candidate library expression in mammaliancells can also be considered for other pharmaceutical-relatedapplications, such as modification of protein expression, proteinfolding, or protein secretion. One such example would be in commercialproduction of protein pharmaceuticals in CHO or other cells. Candidatelibraries resulting in bioactive agents which select for an increasedcell growth rate (perhaps peptides mimicking growth factors or acting asagonists of growth factor signal transduction pathways), for pathogenresistance (see previous section), for lack of sialylation orglycosylation (by blocking glycotransferases or rerouting trafficking ofthe protein in the cell), for allowing growth on autoclaved media, orfor growth in serum free media, would all increase productivity anddecrease costs in the production of protein pharmaceuticals.

[0289] Random peptides displayed on the surface of circulating cells canbe used as tools to identify organ, tissue, and cell specific peptidetargeting sequences. Any cell introduced into the bloodstream of ananimal expressing a library targeted to the cell surface can be selectedfor specific organ and tissue targeting. The bioactive agent sequenceidentified can then be coupled to an antibody, enzyme, drug, imagingagent or substance for which organ targeting is desired.

[0290] Other agents which may be selected using the present inventioninclude: 1) agents which block the activity of transcription factors,using cell lines with reporter genes; 2) agents which block theinteraction of two known proteins in cells, using the absence of normalcellular functions, the mammalian two hybrid system or fluorescenceresonance energy transfer mechanisms for detection; and 3) agents may beidentified by tethering a random peptide to a protein binding region toallow interactions with molecules sterically close, i.e., within asignaling pathway, to localize the effects to a functional area ofinterest.

[0291] The following examples serve to more fully describe the manner ofusing the above-described invention, as well as to set forth the bestmodes contemplated for carrying out various aspects of the invention. Itis understood that these examples in no way serve to limit the truescope of this invention, but rather are presented for illustrativepurposes. All references cited herein are incorporated by reference intheir entirety.

EXAMPLES Example 1

[0292] Construction of BH-1-4 and BH2-A5 cell lines for use in methodsfor screening for an altered cellular phenotype.

[0293] Cell line BH-1-4 The novel cell line, BH-1-4 was constructed byengineering the B cell line BJAB to contain the cDNA encoding thereporter protein heparin-binding epidermal-growth-factor-like growthfactor (HBEGF) operably linked to an interleukin 4 (IL4) induciblegermline promoter, the epsilon promoter (Pε) (see FIG. 1). The epsilonpromoter was activated by contacting the cells with the stimulator IL4.In this cell line, IL4 binds to IL4 receptors (IL4R) expressed on thecell surface and the IL4R mediates transduction and activation of theepsilon promoter by IL4.

[0294] HBEGF functions as the receptor for diphtheria toxin (“dip” or“dt”). Diphtheria toxin is a 535 amino acid protein consisting of threedomains. The receptor binding domain (residues 387-535) binds to BEGF.The transmembrane domain, T, comprising residues 200-378, is responsiblefor creating a channel in the cell membrane. The catalytic domain,comprising residues 1-188, is responsible for stopping potein productionin the cell nd thereby causing cell death. Thus, in the novel BH-1-4cell line, HBEGF confers high sensitivity to the killing of cells bydiphtheria toxin following the induction of the expression of HBEGF bythe addition of the stimulator IL4. Thus, in this example cell death isa marker for the expression of the reporter HBEGF in the presence ofdiphtheria toxin.

[0295] Cell line BH2-A5

[0296] The novel cell line, BH2-A5 was constructed in the same manner asdescribed above for the BH1-4 cell line, except that the DNA encodes thereporter protein Green Fluorescent Protein (GFP) operably linked toHBEGF via a cleavable peptide linker (2 a) (see FIG. 2). Basically, theBH2-A5 cell line was constructed by engineering the B cell line BJAB tocontain the DNA encoding the reporter (HBEGF) operably linked to aninterleukin 4 (IL4) inducible germlne promoter, the epsilon promoter(Pe), and the GFP reporter, where the HBEGF and GFP are linked by thecleavable peptide linker 2 a. Similar to the BH1-4 cell line, theepsilon promoter was activated by contacting the cells with thestimulator IL4; IL4 binds to IL4 receptors (IL4R) expressed on the cellsurface; and the IL4R mediates transduction and activation of theepsilon promoter by IL4. Also, in this cell line, HBEGF functions as thereceptor for diphtheria . Thus, in the novel BH2-A5 cell line, HBEGFconfers high sensitivity to the killing of cells by diphtheria toxinfollowing the induction of the expression of HBEGF by the addition ofthe stimulator IL4. In addition, the concomitant expression of GFP wasused to monitor IL4 induction of the epsilon promoter by fluorescenceusing FACS. Thus, the BH2-A5 cell line was engineered to contain theHBEGF and GFP dual-function reporter. In this example cell death is amarker for the expression of the reporter HBEGF in the presence ofdiphtheria toxin, whereas GFP is a marker for IL4 induced expression.

[0297] Thus, the BH1-4 and BH2-A5 were used to screen for an alteredcellular phenotype due to the presence of a candidate bioactive agent.In this example, the candidate bioactive agent is a member of a peptidelibrary (with a complexity of 1×10e9), where the peptide is operablylinked to a GFP reporter; the parent phenotype is the result ofcontacting the cells with the stimulator IL4 and induction of expressionof HBEGF; and the altered cellular phenotype is cell survival in thepresence of diphtheria toxin in cells expressing HBEGF, where thealtered cellular phenotype is due to the presence of a bioactive agentthat inhibits the IL4-induced expression of HBEGF and thereby results inthe survival of the cells in the presence of diphtheria toxin.

[0298]FIG. 3 depicts the conventional screening method which involvedfirst infecting the cell lines with retroviral vectors encoding acandidate peptide operably linked to a GFP reporter and therebyexpressing the peptide in the cells; culturing the infected cells in thepresence of the stimulator IL4 to induce expression of HBEGF; contactingthe cells with diphtheria toxin to kill cells expressing HBEGF (i.e.,selecting the cells in the presence of diphtheria toxin); removing theIL4 and diphtheria toxin by washing the cells, concentrating them, andremoving the dead cells (“debris”); from the surviving (selected) cells,rescue the retroviral population and infect naïve cells for a new freshround of selection and perform clonal selection and analysis of thecloned retroviral population. However, this step requires that thecomplexity and representation of the population should be maintained asthey are infected into naive cells; and, further, this step istechnically challenging. Thus, the present invention provides a novelapproach that obviates these problems. The following examples furtherillustrate this novel approach for screening for altered cellularphenotypes.

Example 2

[0299] Screening for an altered cellular phenotype using known bioactiveagents, SOCS1 and STAT6Δ (a C-terminal truncated version of STAT6), aspositive controls for the method of screening.

[0300] STAT6 (signal transducer and activator of transcription 6)mediates the response of cytokines like IL4 and SOCS1 (suppressor ofcytokine signaling) and STAT6? Are known inhibitors of IL4 signaltransduction. In the BH1-4 or BH2-A5 cells lines of the presentinvention, SOCS1 and STAT6Δ each inhibit the IL4 inducible expression ofHBEGF (and SOCS1 is a stronger inhibitor than STAT6Δ).

[0301] As depicted in FIG. 4, the following five different retroviralvectors were constructed as positive controls for the screening assay,and are the encoded GFP, SOCS1, STAT6Δ, and/or ires are operably linkedto a promoter in the retroviral vector: the retroviral vector cGFPcontains the GFP; the retroviral vector SOCS1-ires-GFP contains, from 5′to 3′ (and operably linked), SOCS1, internal ribosomal entry site(IRES), and GFP; the retroviral vector GFP-SOCS1 contains, from 5′ to 3′(and operably linked), GFP-SOCS1 fusion; the retroviral vectorSTAT6Δ-ires-GFP contains, from 5′ to 3′ (and operably linked), STAT6Δ,ires, and GFP; and the retroviral vector GFP-STAT6Δ contains, from 5′ to3′ (and operably linked), GFP-STAT6Δ fusion.

[0302] As depicted in FIG. 5 the five retroviral vectors described abovewere assayed in BH1-4 cells using the following protocol: BH1-4 cellswere infected with the retroviral constructs and cultured for 3 days inmedium containing IL4 and diphtheria toxin; or alternatively, theinfected cells were cultured in IL4 for 2 days followed by the additionof diphtheria toxin and culturing in the presence of IL4 and diphtheria.Three days after infections, a 1:10 dilution of the cell cultures wasmade with fresh medium (without IL4 and diphtheria toxin). On the day ofinfection, day 3, day 6 and day 7 GFP fluorescence of the cells wasmeasured using FACS.

[0303]FIG. 6 depicts the effects of SOCS1 and STAT6 on IL4/diphtheria(IL4/dip) induced death of BH1-4 cells 6 days post infection for each ofthe five retroviral vectors, where the histogram panels under the columnentitled “unstimulated” refers to a control assay where the cells werenot stimulated/cultured with IL4; the column entitled “IL4” refers to acontrol assay where the cells were stimulated with IL4 but diphtheriatoxin was not added to the medium; the column entitled “IL4/dip” refersto the assay where the cells were cultured in both IL4 and dip togetherfor 3 days; and the column entitled “IL4 then dip” refers to the assaywhere the cells were cultured in IL4 for 2 days followed by the additionof diphtheria toxin. The histogram in each panel summarizes the FACSmeasurements for each assay. The histograms in the panels for thecontrol assays entitled “unstimulated,” “IL4,” and “Dip” depict thefluorescent scattering pattern indicative of live or surviving cells,whereas the histograms in the panels for the assays entitled “IL4/dip”and “IL4 then dip” for retroviral vectors cGFP, STAT6-IRES-GFP, andGFP-STAT6 depict a the fluorescent scattering pattern indicative of deador killed cells. In contrast, the histograms in the panels for the sameassays, i.e., “IL4/dip” and “IL4 then dip,” for retroviral vectorsSOCS1-IRES-GFP and GFP-SOCS1 depict a fluorescent scattering patternindicative of live cells. FIG. 7 depicts the same results except thatthe vertical axis indicates the number of cells, and the horizontal axisindicates the amount of GFP fluorescence.

[0304] These results indicate that the expression of SOCS1-ires-GFP andGFP-SOCS1 retroviral constructs inhibit IL4 signaling/induced expressionof HBEGF. Further, the results indicate that the GFP report protein isan effective marker for monitoring SOCS1 expression in the screeningassays of the present invention. Finally, these results indicate thatthe expression of GFP-STAT6Δ only partially inhibits IL4signaling/induced expression of HBEGF.

Example 3

[0305] Enrichment of inhibitors of IL4 signaling in BH1-4 or BH2-A5 celllines using SOCS1.

[0306] To examine the degree of enrichment that could be achieved by theabove-described IL4-diphtheria selection assay, BH1-4 and BH2-A5 cellpopulations were “spiked” with BH1-4 and BH2-A5 cells that had beeninfected with the retroviral construct SOCS1-ires-GFP, and the followingdilutions of the SOCS-infected cells with uninfected cells (i.e., BH2-A5and BH1-4 cells not infected with a SOCS1 retroviral construct) weremade:

[0307] 1:10

[0308] 1:100

[0309] 1:1000

[0310] 1:10,000.

[0311] As depicted in FIG. 8, the diluted spiked cell populations werethen subjected to the following procedure: On the day of infection,12×10e6 cells at 200k/ml were stimulated with IL4 (at 30 units/ml) in aT175 flask; On day 1 post infection, diphtheria toxin (dip toxin) at 20ng/ml was added to the cell culture (but prior to the addition of diptoxin, an aliquot of cells was taken as a “no dip control”); On day 3post infection, the cell culture was washed (“washout”) from IL4 anddiphtheria toxin and the cells were grown in regular media; On days 4,6, and 8 post infection, the GFP fluorescence of the cells were measuredusing FACS; On day 9 post infection, the “washout” dying cells weresubjected to ficoll treatment to recover lice cells from dead cells; andOn days 15 and 18 post infection, the GFP fluorescence of the cells weremeasured using FACS.

[0312] The results of this experiment indicate that the screening assayusing L4-dip selection is validated. In particular, the “washout”treated cells ere enriched for the cells having the SOCS1 phenotype byapproximately 1000 fold at the 1:10,000 seeding, from 0.01% to 10%. FIG.9 schematically depicts the IL4-dip selection assay using BH1-4 cellsand depicts the results of the assay starting with a 1:10 dilution ofthe SOCS1-infected cells. The results are depicted in histograms wherethe vertical axis indicates the number of cells and the horizontal axisindicates the amount of GFP fluorescence measured using FACS, the day ofinfection (day 1) and on days 4, 6, and 8 post-infection. GFPfluorescence was used to monitor SOCS1 expression. SOCS1 inhibitsIL4-induced HBEGF expression making the cells expressing it resistant tocell death by IL4-dip treatment. The results indicate that by day 8 themajority of the surviving cells are SOCS1-ires-GFP expressing cells.FIG. 10 depicts the results for the assays using BH1-4 cells startingwith the 1:10, 1:100, 1:1,000, and 1:10,000 dilution of theSOCS1-infected cells. The results are depicted in histograms where thevertical axis indicates the number of cells and the horizontal axisindicates the amount of GFP fluorescence measured using FACS, the day ofinfection (day 1) and on days 4, 6, and 8 post-infection. FIG. 11depicts the results for both BH1-4 and BH2-A5 cells starting with the1:10, 1:100, 1:1,000, and 1:10,000 dilution of the spiked cells. Theresults are depicted in histograms where the vertical axis indicates thenumber of cells and the horizontal axis indicates the amount of GFPfluorescence measured using FACS on day 15 post-infection.

[0313]FIG. 12 depicts the results of selection beginning with naivecells in a first round of selection with IL4-Dip and then subjecting thesurviving cells from the first round of selection to a second round ofselection with IL4-Dip. The naïve cells are the spiked cells diluted1:10 and 1:10,000 with unspiked cells. The first round of selection wasperformed as described above and the amount of fluorescence was measuredusing FACS on days 1, 4, and 18 post infection. For the 1:10,000seeding, the surviving cells selected from the first round weresubjected to a second round of selection performed in the same manner asthe first round, and the amount for fluorescence was measured using FACSon the day IL4 was added to the medium to stimulate IL4 inducedexpression of HBEGF; and on days 4 and 8 thereafter.

[0314] The results of these SOCS1-infected cells spiking experimentsdemonstrate that the IL4-Dip selection in the BH1-4 and BH2-A5 celllines result in enrichment of inhibitors of IL4 signaling/inducedexpression of HBEGF. At a 1:10,000 dilution of the spiked cells, theenrichment by the IL4-Dip selection is approximately 1000 fold (i.e.,from 0.01% to 10%). However, in these experiments, the results indicatethat most survivors of the first selection did not respond to a secondround of IL4 stimulation. Thus, in this case a second round of selectionlead to very little enrichment for the peptide inhibitors. Theconventional or usual solution following selection is to rescue theretroviral population and infect naive cells for a new and fresh roundof selection. However, this step requires that the complexity andrepresentation of the population should be maintained as they areinfected into naïve cells; this requirement makes this step technicallychallenging. Thus, the present invention provides a novel approach thatobviates these problems, as described in the following examples.

Example 4

[0315] Screening for an altered cellular phenotype using aTet-regulatable expression system.

[0316] This example demonstrates a method of screening for an alteredcellular phenotype where cells responsive to stimulation by IL4 (“IL4responders”) are separated from the population of cells that haveundergone the first selection. These IL4 responders can then undergo anadditional round of highly efficient selection by repressing (or“turning down”) the expression of the peptide inhibitors of IL4signaling using a Tet-regulatable (or “Tet inducible”) expressionsystem; and then sorting for the IL4 responders using a reporterprotein, e.g., Green Fluorescent Protein (“GFP”).

[0317] As depicted in FIG. 13, the screening assay in this exampleinvolves infecting. A novel cell line BH2-A5T (described below) with thepeptide library BFP-C20 encoded by retroviral vectors (described below).Thereafter, the cells are stimulated with IL4, selected on IL4-dip, andthen the cells are washed and the surviving cells recovered. The peptideencoded by the retroviral construct is expressed in the absence of Dox(a Tet analogue) and repressed in the presence of Dox. After the firstround of IL4-dip selection, Dox is added to the cell culture mediumcontaining the surviving cells and expression of the peptide is turnedoff. The cells are then stimulated with IL4 to induce expression ofHBEGF-2a-GFP and thereafter sorted for GFP fluorescence (indicative ofIL4 induced expression) using FACS. Thereafter, expression of thepeptide is turned on by removing the Dox. The cells are then selected onIL4-Dip, washed, aliquoted into microtiter plates, sorted by FACS forsingle cell clones, replica plated in duplicate microtiter plates andcultured in the presence or absence of Dox, and the single clones areselected on the basis that the IL-4 induced GFP is inhibited by thepresence of a BFP-peptide as measured by the BFP and GFP fluorescenceusing FACS.

[0318] As depicted in FIG. 14, the BH2-A5 cell line was furtherengineered to construct the cell line BH2A5T (or “A5T” or “A5T-4”) whichexpresses a Tet regulated transactivator (tTA or Tet transactivator) andallows for the Tet regulated expression of candidate bioactive agentsintroduced into the cells. As in the BH2-A5 cells, the BH2-A5T cellscontain the HBEGF-2a-GFP construct that is driven by the epsilonpromoter and is expression of HBEGF-2a-GFP is inducible by stimulationwith IL4. In this example, the candidate bioactive agents are from alibrary of random sequence peptides, 20 amino acids in length, which aremembers of the BFP-C20mer peptide library. The 20 mer peptides areexpressed as carboxy-terminal peptides fused to the reporter protein,Blue Fluorescent Protein (BFP). These fusion peptides (BFP-C20merlibrary member) are operably linked to an oligomer of a Tet operatorsequence (TRA or TetO sequence) and promoter as depicted in FIG. 14. Inthe absence of Tet or analogue thereof (e.g., Dox) the peptide isexpressed in the BH2-A5T cells, whereas in the presence of Tet oranalogue thereof, the expression of the peptide is repressed (or turnedoff).

[0319]FIG. 15 depicts histograms indicating the amount of GFPfluorescence, in an experiment where IL4responders were selected in thepresence and absence of Dox in BH2-A5T cells spiked with cells infectedwith the retroviral construct TRA-SOCS1-ires-GFP. This constructcontains the SOCS1-ires-GFP as described above and in addition isoperably linked to a TRA and thus is regulatable by the tTA. In thisexperiment, the spiked cells were selected for over 7 days on IL4 andDIP, and 1) in the absence of Dox (“IL4/dip selection”); and 2) in thepresence of Dox (“IL4/dip selections+Dox”). After selection, the cellswere cultured in the absence of Dox to allow for the SOCS1-ires-GFPexpression. In summary, the BH2-A5T cell line (expressing tTA) wasgenerated and quality controlled for Dox responsiveness, infectability,responsiveness to IL4 stimulation/induction of expression, and IL4/Dipinduced cell death. These experiments established the fidelity of Doxregulation (turning peptide expression on and off) and kinetics. Theresults demonstrate that peptide expression levels can be 10-17 foldhigher using a TRA containing vector in comparison to the LTR vectorsdescribed above. Further, results from the IL4/Dip selection in thepresence or absence of Dox demonstrate the correlation of enrichment andSOCS1 expression, i.e., enrichment was regulated by the presence orabsence of Dox.

Example 5

[0320] Screening for cells having an altered cellular phenotype bymultiple rounds of sorting of cells responsive to IL4 induced expressionof HBEGF-2a-GFP using an expression system regulatable by Dox, asdepicted in FIG. 13 and further described above in Example 4.

[0321] As described above in Example 3, FIG. 12 depicts the results of afirst round and second round of selection for cells responsive to IL4induced expression, showing that most cells surviving selection fail torespond to IL4 due to the presence of false positives (i.e., stochasticor hereditary nonresponders). The following experiments illustrate byexample how the methods of the present invention solve these problems bythe round to round enrichment for cells expressing the peptide inhibitorusing a combination of 1) induction and repression of the expression ofthe putative peptide inhibitor (or other candidate bioactive agent) viaDox with 2) selections for inhibition or activation of the parentalphenotype.

[0322]FIG. 16 depicts the round to round enrichment for cells expressingthe known inhibitor SOCS1 by induction and repression ofTRA-SOCS1-ires-GFP expression in BH2-A5T-4 cells using dox and sortingof the altered and parental cellular phenotype. In this system, theexpression of TRA-SOCS1-ires-GFP is turned off in the presence of Dox;and SOCS1-ires-GFP is expressed in the absence of Dox. In theseexperiments, BH2-A5T4 cells are infected with the retroviral constructTRA-SOCS1-ires-GFP (as described above); and the infected cells are thendiluted at 1:10,000 and 1:100,000 with uninfected naive cells. In thefirst round of selection (Round 1) the cells are selected on IL4-Dip forthe altered phenotype (as described above); the cells are then contactedwith Dox to repress (or turn off) expression of TRA-SOCS1-ires-GFP andthe cells are stimulated with IL4 and sorted for the parental phenotypeusing GFP fluorescence which is indicative of IL4 induced expression ofHBEGF-2a-GFP. Thereafter, the cells responsive to IL4 induced expressionare collected and cultured in medium without Dox in order to induce (orturn on) the expression of SOCS1-ires-GFP. The cells expressingTRA-SOCS1-ires-GFP are then subjected to a second round of selection forthe altered phenotype on IL4-Dip achieving further efficient enrichmentof SOCS1-expressing cells. FIGS. 17 and 18 show the results of theseexperiments.

[0323]FIG. 17 depicts histograms indicating the amount of GFPfluorescence indicative of TRA-SOCS1-ires-GFP expression in cells afterthe first round of selection. The column entitled “no selection” is acontrol where IL4 and Dip were not added to the cells; the columnentitled “post selection” is a control where the cells were selected onIL4-Dip but were not cultured in the presence of Dox; The results of thefirst round selection are shown for both the selection starting with a1:10,000 dilution of the spiked cells (“TRA-SOCS1-ires-GFP 1/10K”) and1:100,000 dilution of the spiked cells (“TRA-SOCS1-ires-GFP 1/100K”).For the selection starting with a 1:10,000 dilution of the spiked cells(“TRA-SOCS1-ires-GFP 1/10K”), the first round of selection resulted inabout 1000-fold enrichment (from ˜0.01% to ˜9.4%) of cells expressingTRA-SOCS1-ires-GFP. For the selection starting with a 1:100,000 dilutionof the spiked cells (“TRA-SOCS1-ires-GFP 1/100K”), the first round ofselection did not result in a statistically measurable enrichment ofcells expressing TRA-SOCS1-ires-GFP.

[0324]FIG. 18 depicts histograms showing the “Sort for IL4 responders”step in FIG. 16. The columns entitled “no selection” and “postselection” are the same as those in FIG. 17; the column “postselection+dox” depicts the turning off the expression ofTRA-SOCS1-ires-GFP by contacting the cells with Dox for over 7 days inthe cells that had been selected on IL4-Dip; the column “post selection+dox+IL4” shows the GFP profile of “post selection+dox” cells afterbeing contacted with IL4 and Dox for 3 days. Since the SOCS1-ires-GFPexpression is repressed by Dox, the GFP expression in this column isindicative of the IL4-induction of HBEGF-2a-GFP (which is the parentalphenotype); thus 23% to 24% of the cells expressing GFP are IL4responders. From these cell populations, the 10% highest expressing GFPcells (labeled Right (R) gate) and the next 10% (labeled Left (L) gate)were collected as depicted in FIG. 18 for subsequent steps shown inFIGS. 19 and 20.

[0325]FIG. 19 depicts histograms showing the “Turn inhibitor expressionback on” step in FIG. 16. The columns entitled “no selection” and “postselection” are the same as those in FIG. 17 and 18; the panels“sorted-left gate” and “sorted-right gate” show histograms of the sortedpopulations from FIG. 18 after they have been cultured over 5 days inthe absence of Dox (referred to as de-doxing). In this case, the GFPexpression is indicative of the cells expressing TRA-SOCS1.-ires-GFP. Asdepicted in FIG. 19, there is a small enrichment of the cells expressingTRA-SOCS1-ires-GFP resulting from the sorting of the Left Gate and RightGate subpopulation of cells cultured in the absence of Dox. FIG. 19depicts for the 1:10,000 dilution of cells, an 8% enrichment resultingfrom the first round of selection, a 16% enrichment from Left Gate cellsand 18% enrichment from Right Gate cells resulting from the sorting ofthe cells cultured in the absence of Dox; and for the 1:100,000 dilutionof cells, an 0.6% enrichment resulting from the first round ofselection, a 0.9% enrichment from Left Gate cells and 1.0% enrichmentfrom Right Gate cells resulting from the sorting of the cells culturedin the absence of Dox. For the next step, these cell populations aresubjected to a second round of IL4/dip selection as summarized in FIG.20.

[0326]FIG. 20 depicts for the 1:10,000 dilution of cells, an 6.1%enrichment resulting from the first round of selection; for the secondround of IL4/dip selection, a 88% enrichment from the de-doxed Left Gatecells and a 97% enrichment from de-doxed Right Gate cells from FIG. 19;and for the 1:100,000 dilution of cells, an 0.3% enrichment resultingfrom the first round of selection; for the second round of IL4/dipselection, a 26% enrichment from the de-doxed Left Gate cells and 47%enrichment from de-doxed Right Gate cells resulting from the sorting ofthe cells cultured in the absence of Dox (FIG. 19).

[0327] A summary of the screening assay in this example is schematicallydepicted in FIG. 20 where in the first round of selection the cells areselected on IL-4-Dip; and then the surviving cells are cultured in thepresence of dox and sorted for responsiveness to IL4 induced expressionof HBEGF-GFP (i.e., sorted for “IL4 responders”). From the population ofIL4 responders, a Left Gate and Right Gate subpopulation of cells iscollected and cultured in the absence of dox (or “dedoxing”). In theabsence of dox, the expression of TRA-SOCS1-ires-GFP is turned back onand subjected to a second round of IL4-Dip selection as described above.

[0328] The results of these experiments demonstrate that cells harboringa petide causing an altered phenotype can be highly enriched andselected for by the methods of the present invention. Specifically, theround to round induction and repression of expression using Dox resultedin the enrichment and selection of cells responsive to IL4 inducedexpression having an altered cellular phenotype due to the presence ofTRA-SOCS1-ires-GFP. Further, in screening experiments, the putativepeptide inhibitor is operably linked to BFP, thus avoiding any potentialconflicts between the sorting for GFP fluorescence indicative ofPε-HBEGF-2a-GFP expression and sorting for BFP fluorescence indicativeof the expression of the putative peptide inhibitor (or candidatebioactive agent).

Example 6

[0329] Methods for screening for an altered cellular phenotype using aTet-regulatable expression system and a screening cell line, e.g.,BH2-A5T.

[0330]FIG. 21 schematically depicts an example of a timeline (in days)for a screening assay of the present invention, where the assay involvesa first round of selection and sorting; a second round of selection andsorting; and thereafter single cell clones are grown. The single cellclones are then subjected to selection and FACS assays, the nucleic acidencoding the bioactive agent (e.g., a peptide inhibitor of IL4signaling/induced expression) is then rescued and the phenotype isreconfirmed, e.g., by infecting naive cells with the rescued nucleicacid and selection.

[0331]FIG. 22 schematically depicts an example of a timeline (in days)for a screening assay of the present invention (and as described forFIG. 21), where the complexity of the library of candidate bioactiveagents (e.g., a peptide library), and the fold enrichment for thealtered cellular phenotype, are indicated. Further, FIG. 22schematically depicts the histogram profile of GFP fluorescence of falsepositives due to hereditable background or stochastic non-hereditablebackground; as compared to the histogram profile of GFP fluorescence ofcells cultured in the presence (+Tet) or absence (−Tet) of Tet, after afirst round of selection.

[0332]FIG. 23 schematically depicts an example of a timeline (in days)for a screening assay of the present invention (and as described forFIG. 21), where the complexity of the library of candidate bioactiveagents (e.g., a peptide library), and the fold enrichment for thealtered cellular phenotype, are indicated. Further, after a second ofselection, cells are single cell cloned, aliquoted into microtiterplates, replica plated in duplicate microtiter plates and cultured inthe presence or absence of Dox, and the single clones are contacted withIL4 for three days and their GFP fluorescence measured by FACS. FIG. 23schematically depicts the histogram profile of GFP fluorescence of falsepositive clones due to hereditable background or stochasticnon-hereditable background; as compared to the histogram profile of GFPfluorescence of cell clones harboring a peptide inhibitor cultured inthe presence of Tet (+Tet) or absence of Tet (−Tet) (see panel ofschematically depicted histograms). FIG. 23 depicts an example of aclone where in the presence of Tet the expression of the peptideinhibitor is turned off and thus the GFP reporter is expressed; and inthe absence of Tet the peptide is expressed and thus the GFP reporter isinhibited in cells having an altered phenotype. Thus, this exampledemonstrates a powerful approach to identifying and eliminating (ordisregarding) background cell clones (e.g., false positives) byselecting only those clones responsive to IL4 stimulation (i.e., IL4induced expression) that is regulated by Tet.

[0333]FIG. 24 depicts the histogram profile of GFP fluorescence ofclones from a functional screen representing a BFP-peptide inhibitorclone, CR2 (left panel); a hereditable background clone (middle panel),and stochastic background clone (right panel), where the histograms fromthe clones cultured in the presence of Dox (+Dox) and the absence of Dox(−Dox) are overlayed. In the presence of Dox the expression of theBFP-peptide is turned off; and in the absence of Dox the BFP-peptide isexpressed.

[0334]FIG. 25 depicts the summary of the results from the peptidescreening in BH2-A5T-4cells. In this screen, the total number of cellstargeted with the retroviral vector peptide library was 1.25×1010. Fromthe total cells targeted, the total number of cells infected with aretroviral member of the library was 2.4×109. From the total cellsinfected, the total number of cells sorted/cloned was 24,960. From thetotal number of cells sorted/cloned, the total number of regulatableclones, was 1,525. FIG. 25 further depicts a protocol for characterizingthe identified regulatable clones by rescuing the retroviral constructencoding the peptide from the clones, cloning and sequencing the nucleicacid encoding the peptide, and testing for the transfer of the alteredcellular phenotype (conferred by the presence of the peptide) into naïvecells.

Example 7

[0335] Identification and validation of novel signaling moleculesspecific for T cell activation and effector function by screening for analtered cellular phenotype in activated T cells.

Abstract

[0336] T lymphocytes play crucial roles in immune responses, includingthe direct killing of virus-infected cells by cytotoxic T cells andfacilitation of B-cell responses by helper T cells. The activation of Tcells is mediated by the T cell receptor (TCR), which in turn activatesspecific membrane-associated and intracellular proteins. Identifyingthese signaling proteins downstream of TCR activation is crucial fordeveloping therapeutic agents to inhibit or regulate immune responses inautoimmune diseases and organ transplantation. Using the methods andcompositions of the present invention novel signaling molecules specificfor T cell activation and effector function were identified andvalidated. A large library (5×10⁷) of cDNA clones were introduced into Tcells from which an altered cellular phenotype was enriched for andidentified. Using the screening methods of the present invention, 2,800individual clones were obtained based on a reduction in T cell receptoractivation-induced CD69 expression, and the causal relationship of cDNAexpression and altered phenotype was established. In addition to manyknown signaling molecules such as LCK, ZAP70, SYK, and PLC(I, moleculespreviously unknown to this pathway were also discovered using thescreening methods of the present invention. When selected for evaluationwith primary human T lymphocytes, hits from the screen inhibitedanti-CD3 and anti-CD28 stimulated IL-2 production. From these molecules,potential therapeutic targets may be identified that are effective inmodulating immune-mediated processes.

Introduction

[0337] Activation of specific signaling pathways in lymphocytesdetermines the quality, magnitude and duration of immune responses. Intransplantation, acute and chronic inflammatory diseases, andautoimmunity, it is these pathways that are responsible for theinduction, maintenance and exacerbation of disease lymphocyte responses.In all cases, recognition of antigens presented by the MajorHistocompatability Complex (MHC) by the T cell receptor (TCR) complextriggers the activation of T lymphocytes. Engagement of the TCR byantigen/MHC results in actin cytoskeleton rearrangement, induction ofcyrokine and other gene transcription, and progression into the cellcycle^(1,2). The proximal events of TCR signaling include activation ofsrc family kinases LCK, FYN, phosphorylation of TCR component (. ) andsubsequent activation of ZAP70/SYK tyrosine kinases, as well asrecruitmentof adaptormolecules(CBL-B, LAT, SLP76), which couple to moredistal signaling pathways including Ras and PLC(³⁻⁵. New components ofthe TCR signaling pathway have been discovered and reported, such as thenew transmembrane adaptor PAG/CBP⁶, albeit with a slower pace. It hasbecome apparent that identifying additional signaling molecules requiresnovel approaches including functional genomics. Using the methods of thepresent invention, novel signaling molecules specific for T cellactivation and effector function were identified and validated.

[0338] In this example, using the methods of the present invention, anovel approach to identifying new targets for immune suppressive drugsis presented. Following T cell activation, expression of numerous cellsurface markers such as CD25, CD69, and CD40L are upregulated. CD69 hasbeen shown to be an early activation marker in T, B, and NK cells⁷⁻⁹.CD69 is a disulphide-linked dimer. The cell surface marker is notexpressed in resting lymphocytes but appears on T, B and NK cells afteractivation in vitro. The relevance of CD69 as a TCR signaling outcomehas been validated using T cells deficient in certain key signalingmolecules such as SLP76 and LAT^(10,11). Furthermore, re-introducingSLP76 or LAT into the deficient cells resulted in restoration of CD69expression. The CD69 upregulation could then be used to monitor TCRsignal transduction. The rationale of the functional genomics screen wasto identify cell clones whose CD69 upregulation was repressed followingintroduction of a retroviral cDNA library. The library membersconferring such repression would then represent immune modulators thatfunction to block TCR signal transduction.

[0339] The use of retrovirus-mediated gene transfer has contributed tothe cloning of T cell antigens¹², tumor antigens³, variousreceptors¹⁴⁻²⁴, signaling molecules²⁵ and transcription factors²⁶. Inmost of the cases, retroviral cDNA libraries were used for expressionalcloning. The unique feature of the current study reported here, is theuse of retroviruses as pharmaceutical tools for target discovery andvalidation a long a whole pathway of signal transduction, fromligand-receptor interaction on the plasma membrane, membrane-proximalsignaling, actin-cytoskeleton rearrangement to gene transcription in thenucleus. Another feature of our screen was to build-in the functionalrelevance through partial and full-length cDNAs in the library.Expression of these library inserts was expected to have dominanteffects over their endogenous counterparts by being competitiveinhibitors of endogenous protein activity, or being constitutivelyactive. Integration of several key technology innovations such as newcell lines, improved retroviral infection efficiency, high level andregulated expression of nucleic acids (e.g., of a nucleic acid library),and high throughput screening tools complemented successful execution ofthese screens.

Results and Discussion

[0340] Experimental design. Several T cell linesj-including Jurkat,HPB-ALL, HSB-2 and PEER were tested for the presence of surface CD3,CD25, CD28, CD40L, CD69, CD95, and CD95L. Those that expressed CD3 werecultured with anti-CD3 or anti-TCR to crosslink the TCR and examined forthe upregulation of CD69. A Jurkat T cell line was selected for itsability to upregulate CD69 in response to TCR crosslinking with kineticsmimicking that of primary T lymphocytes (data not shown). The populationof Jurkat cells was sorted for low basal and highly inducible CD69expression following anti-TCR stimulation.C lone 4D9 was selectedbecause CD69 in this clone was uniformly and strongly induced followingTCR stimulation in 24 hours (FIG. 26A).

[0341] In order to regulate the expression of the retroviral library,the Tet-Off system was used. The cDNA inserts in the retroviral librarywere cloned in a manner to operably link the inserts to the tetracyclineregulatory element (TRE) and the minimal promoter of TK. Transcriptionof the cDNA inserts was then dependent on the presence oftetracycline-controlled trans-activator (tTA)²⁷, a fusion of Tetrepression protein and the VP16 activation domain, and the absence oftetracycline or its derivatives such as doxycycline (Dox). To shut offthe cDNA expression, one can simply add doxycycline in the medium. Toobtain a Jurkat clone that stably expresses tTA, a retroviral LTR-driventTA in conjunction with a TRE-dependent reporter constructwasintroduced, i.e., TRA-Lyt2. Through sorting of Lyt2 positivecells in theabsence of Dox and Lyt2 negative cells in the presence of Dox, coupledwith clonal evaluation, we obtained a derivative of Jurkat clone 4D9,called 4D9#32, that showed the best Dox regulation of Lyt2 expression,as seen in FIG. 26B.

[0342] Positive controls. ZAP70 is a positive regulator of T cellactivation²⁸. A kinase-inactive (KI) ZAP70 and a truncated ZAP70 (SH2N+C)²⁹ were subcloned into the retroviral vector underTRE control (FIG.27A). As seen in FIG. 27B, ZAP70 SH2 (N+C) and ZAP70 KI both inhibitedTCR-induced CD69 expression. Consistent with the published report ofdominant negative forms of ZAP70 on NFAT activity 29, the truncatedprotein is also a more potent inhibitor of CD69 induction compared tothe K protein, which had a single point mutation in the catalyticdomain. In addition, with higher protein expression, as measured byhigher levels of GFP from the bi-cistronic ZAP70 SH2 (N+C)-IRES-GFP andZAP70 KI-IRES-GFP constructs, inhibition of CD69 induction was stronger(data not shown).

[0343] The CD69 inhibitory phenotype is dependent on expression ofdominant negative forms of ZAP70. As shown in FIG. 27C, when Dox wasadded before TCR was stimulated, there was no inhibition of CD69expression. FACS analysis of cellular GFP expression revealed a lack ofGFP+cells, supporting the notion that the bi-cistronic ZAP70 SH2(N+C)-IRES-GFP mRNA was not transcribed. The lack of ZAP70 SH2 (N+C)protein expression in the presence of Dox was confirmed by Western (FIG.27D). In the absence of Dox, each cell population contained roughly 50%of GFP—cells that did not express the retrovirally introduced ZAP70variants. Thus, FIG. 27D showed that it took on average 4-5 fold higherlevel of ZAP70 SH2 (N+C) than the endogenous ZAP70 to achieve adominant-negative effect.

[0344] Screening for cells lacking CD69 upregulation. FIG. 28A diagramsthe scheme to obtain cell clones with CD69 inhibitory phenotype. Jurkat4D9#32 cells were infected with cDNA libraries made form primary humanlymphoid organs such as thymus, spleen, lymph node and bone marrow. Thelibrary complexity was 5×10⁷ and was built on the TRE vector. Theinfection rate was 52%, as judged by infection with TRA-dsGFP inparallel experiment using the same host cells (data not shown).TRA-dsGFP was another TRE-dependent reporter construct using the samevector backbone as that used by the cDNA libraries or TRALyt2. Afterlibrary infection, cells were stimulated with the antiTCR antibody C305overnight. A total of 7.1×10⁸ cells were stained with anti-CD69 antibodyconjugated to allophycocyanin (APC) and anti-CD3 antibody conjugated tophycoerythrin (PE) and screened using flow cytometry. Even though therewas a significant reduction of CD3/TCR complex on the surface due toreceptor-mediated internalization, compared to unstimulated cells (datanot shown), the CD3-population was distinguishable from theCD3+population. Greater than 2% cells lacking the CD3-PE staining (CD3−)(>2% of total cells had lost TCR/CD3 complex) were consistentlyobserved, which explained their unresponsiveness to stimulation and,consequently, low CD69 expression. Based on this fact, only cells withthe lowest CD69 expression which still retained the CD3 expression wereselected. The desired altered phenotype was termed CD69^(low) CD3⁺ (FIG.28A), which represented 1% of the total stained cells (FIG. 28B). The 1%sorting gate also translated as 100-fold enrichment in the first roundof sorting alone, based on cell numbers. The recovered cells wereallowed to rest in complete medium for 5 days before being stimulatedagain for a new round of sorting. In subsequent rounds of sorting, thesorting gate was always maintained to contain the equivalent of 1% ofthe control cells that were stimulated but were never flow-sorted. Asshown in FIG. 28B, an enrichment was achieved after 3 rounds ofreiterative sorting. Cells with the desired CD69^(low) CD3⁺ phenotypeincreased from 1% to 23.2%. In addition, the overall population'sgeometric mean for the CD69 fluorescent intensity was also reduced(from >300 to 65).

[0345] In order to ascertain that the phenotype was due to expression ofthe cDNA library rather than to spontaneous or retroviralinsertion-mediated somatic mutation, the cells recovered after the thirdround of sorting were split into two populations. One half of the cellswere grown in the absence of Dox while the other half in the presence ofDox for 6 days. CD69 expression was analyzed following anti-TCRstimulation overnight.

[0346] As shown in FIG. 28C, cells with the CD69^(low) CD3⁺ pherotypedecreased from 24.0% to 13.0% with the addition of Dox, demonstratingthat a significant number of cells (11%) had lost the CD69^(low) CD3⁺phenotype in the presence of Dox. These data suggested that theCD69^(low) CD3⁺ phenotypein a significant number of cells in thepopulation was indeed caused by the expression of the cDNA librarymembers. Single cell clones were deposited in conjunction with thefourth round of CD69^(low) CD3⁺ sorting. The cells from threeconsecutive rounds of CD69^(low) CD3⁺ sorting were referred to as LLL,and those from four consecutive rounds of CD69^(low) CD3⁺ sorting werereferred to as LLLL. Overall, cells from up to 7 rounds of sorting(LLLLLLL) were collected.

[0347] In order to reduce the number of cells whose phenotype was notDox-regulatable, the population of the cells grown in the presence ofDox were subjected to a different round of sorting. The purpose for thisround of sorting was to enrich for cells having normal CD69 expressionwhen the library cDNA expression was switched off in the presence ofDox. This sorting variation was termed LLLH where H means CD69^(high).The cells recovered from LLLH sort were cultured in the absence of Doxfor subsequence sorting of the CD69^(low) CD3⁺ phenotype. Single cellclones with the CD69^(low) CD3⁺ phenotype were deposited in 96-wellplates from both the LLLHL and LLLL sorting variations. Table 1 showedthat indeed a higher percentage of Dox-regulatable clones with the LLLHLsorting variation than with LLLL was achieved. Functional Analysis ofSingle Cell Clones. After single cell clones started to grow intoindividual colonies in 96-well plates, their cellular phenotype wascharacterized with the cDNA expression turned on or off, by growing thecells in the presence or absence of Doxcycline (Dox). FIG. 29A showssome examples of the Dox-regulatable phenotypes for individual clones.Dox regulation of CD69 expression was expressed as the ratio ofgeometric mean fluorescent intensity (GMFI) in the presenceof Doxdivided by the CD69 GMFI in the absence of Dox after TCR stimulation.This ratio of (+Dox)/GMFI (−Dox) was termed the Dox ratio. In uninfectedcells, Dox had little or no effect on the induction of CD69 expressionso that the Dox ratio for individual clones on all occasions wasconsistently 1.00+/−0.25 (standard deviation). Therefore, the 2xstandard deviation was used as a cut-off criterion and designated cloneswith a ratio above 1.5 as Dox regulated clones. Out of 2828 clonesanalyzed, 1323 had a Dox-regulatable cellular phenotype as judged by theabove criteria, representing 46.8% of analyzed clones. FIG. 29B showsthe distribution of clones with the Dox ratio between 1.5 and 10, whichcontained 1186 clones. Interestingly, the majority of clones have a Doxratio below 10 whereas rare clones were discovered with a Dox ratio upto 70. RNA samples were prepared from clones with Dox-regulatablephenotypes. Using primers specific for the vector sequence flanking thecDNA library insert, we captured the cDNA insert of selected clones byRT-PCR. FIG. 29C showed the pattern of RT-PCR products. Most clonesgenerated only one DNA band, whereas a few clones generated two or morebands. Sequencing analysis revealed that the additional bands wereusually caused by double or multiple insertions of retroviruses.Occasionally, the two PCR products in the same lane representeddifferent fragments of the same gene product. The results of the cDNAanalysis are summarized in Table 2.

[0348] Characterization of proteins critical for T cell activation. Asshown in Table 2, known TCR regulators such as LCK. ZAP70, SYK, andPLC(1 were obtained using the methods of the present invention.

[0349] LCK is a non-receptor protein tyrosine kinase^(30,31). Its rolein T cell development and activation has been widely documented 3234 Todate, dominant negative forms of LCK have not been reported. Thediscovery by the present inventors that over expression of thekinase-truncated form of LCK caused inhibition of CD69, similar to thephenotype of Jurkat somatic mutant lacking LCK 35, suggests that kinasedeletion of LCK could also work as a dominant negative form of LCK (FIG.30A).

[0350] The two ZAP70 hits both contained the endogenous ATG initiationcodon and ended at aa 262 and 269, respectively (FIG. 30B). They bothare missing the catalytic domain. The deletions are very close to thepositive control for the screen, ZAP70 SH2 (N+C), which ended at aa276²⁹. Since ZAP70 SH2 (N+C) was shown to be a dominant negative protein29 (also see FIG. 27), the two ZAP70 hits are believed to also behave asdominant negative proteins of ZAP70 (FIG. 30B).

[0351] SYK is a non-receptor tyrosine kinase belonging to the SYK/ZAP70family of kineses³⁶. Since it has also been shown that the lack of SYKexpression in Jurkat cells did not appear to significantly alter theTCR-mediated responses compared with Jurkat clones expressing SYK³⁷, theSYK hit obtained from the present screen is believed to function mainlyin blocking ZAP70 function (FIG. 30C and data not shown). SYK'ssimilarity to ZAP70, its ability to associate with phosphorylated TCR;chain and its ability to reconstitute the ZAP70-deficient Jurkat T-cellline also support this notion³⁸.

[0352] PLC(I plays a crucial role in coupling T cell receptor ligationto IL-2 gene expression in activated T lymphocytes¹. TCR engagementleads to rapid tyrosine phosphorylation and activation of PLC(I³⁹. Theactivated enzyme converts phosphatidylinositol-4, 5-bisphosphate (PIP2)to inositol-1, 3,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3triggers intracellular Ca²⁺ increase and DAG is a potent activator ofprotein kinase C (PKC). PLC(1 has a split catalytic domain comprised ofconserved X and Y subdomains (FIG. 30D). Single point mutation in thecatalytic X box completely abolished the enzyme activity and alsoblocked IL-2 reporter gene expression when introduced intoPLC(I-deficient Jurkat cells⁴⁰. The hit contained the PH domain and theN and C terminal SH2 domains of PLC(I (FIG. 30D). Significantly this hitalso deleted the crucial tyrosine Y783 between the SH2 (C) and SH3domains. It was reported that Y783 was essential for coupling of TCRstimulation to IL-2 promoter activation and that mutation of Y783 to F(phenoalanine) generated a very potent dominant negative form ofPLC(I⁴⁰. Indeed, the original clone encoding the PLC(I hit had thehighest Dox ratio for CD69 expression among all clones from the cDNAscreen, indicating the strong repression of CD69 induction by the PLC(1hit as well as the total de-repression in the absence of the hitexpression. When introduced to naive Jurkat cells, this fragment causedsevere block of TCR-induced CD69 expression (FIG. 30D).

[0353] Other signaling molecules known to involve in TCR signalingpathway were also discovered using the methods of the present invention.They included PAG⁶, CSK^(41,42,43), SHP-1⁴⁴ and nucleolin⁴⁵ (Table 2 anddata not shown). Raf is a MAP kinase kinase kinase. It interacts withRas and leads to activation of the MAP kinase pathway⁴⁶. Raf- 1 wasreported to participate in TCR signaling^(47,48). The Raf hit obtainedcorresponds to the truncated form of A-raf, missing the kinase domain.It is likely that this A-raf hit is a dominant negative form of the Raf-1 kinase^(49,50). Alternatively, our result suggests that A-Raf is alsocritical for TCR signaling leading to CD69 activation.

[0354] In addition to the known signaling molecules, using the methodsof the present invention the function of genes whose identity werereported previously, but whose involvement in TCR signaling was notdocumented, was discovered. For example, SH2-B was originally identifiedby its ability to associate with the immunoreceptor tyrosine-basedactivation motif (ITAM) of the FcgR (chain⁵¹. It belongs to asuperfamily of intracellular signaling molecules including LNK andAPS^(52,53). Recently, LNK was shown to be crucial for B cell production54. It is conceivable that SH2-B plays an analogous immune modulatoryrole in T cell activation (Table 2).

[0355] TCPTP (T cell protein tyrosine phosphatase) is also called PTPN2for protein tyrosine phosphatase nonreceptor-2⁵⁵. It was reported to behighly expressed in T cells but its function in TCR signal transductionwas not elucidated. The hit obtained from the using the screeningmethods of the present invention contained a C-terminal truncation andan intact phosphatase domain (FIG. 31A). Constitutively active TCPTP wasreported to have a C-terminal truncation due to protease cleavage at aa.387⁵⁶. Interestingly, the hit ends at aa 369, just 18 aa shorter thanthe protease cleavage product. It is conceivable that the hit is aconstitutively activate TCPTP and that activated TCPTP is a negativeregulator of TCR signaling.

[0356] IL-10 receptor⁵⁷ and Integrin “2⁵⁸ are both transmembraneproteins. The cytoplasmic regions of these two receptors were identifiedas inhibitors of TCR-induced CD69 expression (FIG. 31B and FIG. 31C),using the methods of the present invention. Although the mechanisms ofthe inhibition were not clear, these transmembrane molecules may serveas specific “sinks” for other positive signaling molecules.Alternatively, a more appealing explanation is that these transmembranemolecules specifically modulate T cell activation. For example,stimulation of T cells via the CD3-TCR complex resulted in rapidincrease in $1 integrin-mediated adhesion. Integrins are capable ofinside-out signal transduction⁵⁹ and have been reported to bind toSLP-76⁶⁰. The hematopoietic-specific adaptor SLAP-130/Fyb was also shownto be important for coupling TCR-mediated actin cytoskeletalrearrangement with activation of integrinfunction, and for T cells torespond fully to activating signals⁶¹. Recently, it has been reportedthat the Tec family tyrosine kinase ITK⁶² regulated the inside-outsignaling events from TCR to integrins⁶³. Furthermore, Integrin “2$1mediates p38” activation and upregulation of collagen gene transcriptionby a mechanism involving the “2 cytoplasmic tail, Cdc42, MKK3 andMKK4⁶⁴. Taken together, our discovery of the dominant negative effect ofthe cytoplasmic tail of” integrin strengthened integrin's role infunctionally modulating T cell activation (FIG. 31C).

[0357] In addition to uncovering truncated cDNAs encoding dominantnegative mutants of the positive regulators and constitutively activemutants of the negative regulators of T cell activation, using thescreening methods of the present invention a clone encoding thefull-length open reading frame of the gene GG2-1 (Table 2 and FIG. 31D)was also identified. GG2-1, also called SCC S2, was independentlydiscovered as a transcript upregulated with TNF”^(65,66). GG21/SCC-S2contained a sequence in the amino terminus that shows a significanthomology to death effector domain 11 of the cell death regulatoryprotein FLIP. Unlike FLIP, the GG2-1/SCC-S2 open reading frame containsonly one death effector domain and lacks the carboxyl-terminalcaspase-like homology domain. The significance of the GG2-1 hit from ourT cell activation screen awaits further studies.

[0358] Function in primary T lymphocytes. The relevance of the cDNAhits, identified by the methods of the present invention, to thephysiological function of T cells was investigated in primary Tlymphocytes. The hits were subcloned into a retroviral vector under aconstitutively active promoter embedded in the retroviral LTR, followedby IRES-GFP. A protocol was developed to couple successful retroviralinfection to subsequence T cell activation. Primary T lymphocytes are atthe quiescent stage when isolated from healthy donors. In order to beinfected by a retrovirus, primary lymphocytes need to be activated toprogress into the cell cycle. As shown in FIG. 32A, fresh peripheralblood lymphocytes (PBL) contained typically T cells and B cells. Thecombined CD4⁺ and CD8⁺ cells represented total T cells, which were 81%in this particular donor. The remaining 19% CD4⁺ and CD8⁺ cells were Bcells as stained by CD19 (data not shown). Upon culturing on anti-CD3and anti-CD28 coated dishes, primary T lymphocytes were expanded andprimary B cells and other cell types gradually died off in the culture.FIG. 32 showed that after infection, the culture contained virtually allT cells. Furthermore, primary T lymphocytes were successfully infectedby retroviruses (FIG. 32A and B). As seen with Jurkat cells (data notshown), GFP translated by way of IRKS was not as abundant as GFPtranslated using the conventional Kozak sequence (comparing GFPgeometric mean from CRU5-IRES-GFP and CRU5-GFP). Nevertheless thepercentage infection remained similar. Insertion of a gene in front ofIRES-GFP further reduced the expression level of GFP, which was observedwith many cell lines (data not shown) and here in primary T lymphocytes(FIG. 32B). After allowing cells to rest following infection, we flowsorted cells into two populations: GFP⁻ and GFP⁺. The sorted cells wereimmediately put into culture. As seen in FIG. 32C, anti-CD3 alone didnot induce IL-2 production. This observation was consistent withprevious report on freshly isolated primary T lymphocytes and confirmedthe notion that prior culture and retroviral infection did not damagethe physiological properties of these primary T lymphocytes. Addition ofanti-CD28 in conjunction with anti-CD3 led to robust IL-2 productionwith vector-infected cells and the GFP⁻ population of LckDN andPLC(IDN-infected cells. The GFP⁺ cell population from LckDN andPLC(IDN-infected cells, however, were severed impaired in IL-2production following anti-CD3 and anti-CD28 stimulation (FIG. 32C). Asexpected, the defect caused by LckDN and PLC(IDN can be completelyrescued by stimulation using PMA and ionomycin (FIG. 32C). Takentogether, these results showed that LCK and PLC(1 play crucial role inIL-2 production from primary T lymphocytes, consistently with theirinvolvement in membrane proximal signaling events of T cell activation.These results also demonstrated a quick system to further validate hitsusing the methods of the present invention as a functional geneticscreen in primary cells.

[0359] In conclusion, the methods of the present invention used in thisstudy demonstrates a successful approach to discover and validate in afunctionally relevant context important immune regulators on agenome-wide scale. This approach, which requires no prior sequenceinformation, provides a tool for functional cloning of regulators innumerous signal transduction pathways. For example, B cellactivation-induced CD69 expression (Holland et al., in press) andIL4-induced IgE class switch are all amendable to the geneticperturbation following introduction of retroviral cDNA libraries. Themethods of the present invention are less biased compared to forcedintroduction of a handful of signaling molecules discovered in othercontexts such as growth factor signal transduction. The presentinvention also open the door for discovering peptide inhibitors ofimmune modulatory proteins by screening random peptide librariesexpressed from retroviral vectors using the screening methods of thepresent invention.

Experimental Protocol

[0360] Preparation of cDNA libraries. mRNA extracted from human lymphnodes, thymus, spleen and bone marrow was used to produce two cDNAlibraries; both randomly primed. One library (−ATG) inserts weredirectionally cloned and the second (+ATG) non-directionally cloned andprovided with 3 exogenous ATG in 3 frames. cDNAs were cloned into thepTRA-exs vector to give rise to robust Doxycycline-regulatabletranscription from the TRE enhancer and the minimal promoter in celllines expressing tTA. The total combined library complexity was 5×107independent clones.

[0361] Cell lines. Phoenix A cells were cultured in DMEM supplementedwith 10% fetal calf serum, penicillin and streptomycin. Human T cellleukemia line Jurkat was obtained from Novartis and was routinelycultured in RPMI 1640 medium supplemented with 10% fetal calf serum,penicillin and streptomycin. To obtain the clone with optimal CD69induction, Jurkat cells were sorted for low basal CD69 expression andhigh induction of CD69 expression following TCR stimulation. To producethe tTA-Jurkat cell line, Jurkat clone (4D9) with optimal CD69expression profile was infected with retroviral construct whichconstitutively expresses the tetracycline transactivator protein (tTA)and a reporter construct which expresses Lyt2 driven by a tetracyclineresponsive element (TRE). The tTA-Jurkat cell clone 4D9#32 was obtainedby sorting for high Lyt2 expression in the absence of Doxycycline andlow expression of Lyt2 in the presence Doxycycline (10 ng/ml).

[0362] Transfection and infection. Phoenix A packaging cells weretransfected with retroviral vectors using calcium phosphate for 6 hoursfollowing standard protocols. After 24 hours, supernatant was replacedwith complete RPMI medium and virus was allowed to accumulate for 24hours at 32EC. Viral supernatant was collected, filtered through a 0.2 Mfilter and mixed with Jurkat cells at a density of 5×10⁵ cells/mi. Cellswere spun at room temperature for 3 hours at 2500 rpm, followed byovernight incubation at 37EC. Transfection and infection efficiencieswere monitored by flow cytometry. Functional analysiswas carried out 2-4days after infection.

[0363] Stimulation. For CD69 upregulation experiment, Jurkat cells weresplit to 2.5×10⁵ cells/ml 24 hours prior to stimulation. Cells were spunand resuspended at 5×10⁵ cells/ml in fresh complete RPMI medium in thepresence of 300 ng/ml C305 (anti-Jurkat clonotypic TCR) hybridomasupernatant for 20-26 hoursat 37EC, and then assayed for surface CD69expression.

[0364] Antibodies and Flow cytometry. Jurkat cells or human peripheralblood lymphocytes were stained with FITC-conjugated monoclonalanti-mouse CD8″ (Lyt2), APC conjugated mouse monoclonal anti-human CD3,anti-human CD8, or anti-human CD69 antibodies, and PE-conjugated mousemonoclonal anti-human CD3 or anti-CD4 antibodies (all from Caltag) at4EC for 20 minutes and analyzed using a FACSCalibur instrument (BectonDickinson) with the CellQuest software. Fluorescent-activated cellsortings were performed on the MoFlo instruments (Cytomation).

[0365] cDNA library screen. Phoenix A packaging cells were transfectedwith a mixture of the two tTA regulated retroviral cDNA libraries (totalcomplexity 5×10⁷). Supernatant containing packaged viral particles wasused to infect 3.5×10⁸ tTA-Jurkat cells with an efficiency of 52% basedon parallel infection with TRA-GFP. After 4 days of cDNA expression,library infected cells were stimulated with 300 ng/ml C305 for 20-30hours, stained with APC-conjugated anti-CD69 and PE-conjugated anti-CD3,and 1% of total cells expressing the lowest CD69 level and stillpositive for CD3 expression were isolated using a fluorescence activatedcell sorter (Cytomation). Sorting was repeated multiple rounds with a6-day rest period between stimulations until the population wassignificantly enriched for the desired phenotype of CD69^(low)CD3⁺.Following 3 consecutive sorting for the CD69^(low) CD3⁺ phenotype afterTCR stimulation in the absence of Dox, half of the cells were culturedand stimulated in the presence of Dox and sorted for the top 10% of theCD69 level (so called CD69^(high) henotype) to enrich for clones whosephenotype was dependent on cDNA expression. Single cells were depositedfrom 8 separate rounds of sorting with different variations of theplacement of the CD69^(high) sort. Cell clones were expanded in thepresence and absence of Dox, stimulated and analyzed for CD69upregulation.

[0366] Isolation of cDNA inserts. PCR primers were designed to amplifycDNA inserts from both libraries and did not amplify Lyt2 that was alsounder TRE regulation. The primers used contained flanking BstXI sitesfor subsequent cloning to the pTRA-IRES-GFP and CRU5-IRES-GFP vectors.BstXTRA5G: 5′TTGCAGMCCACCACCTTGGGCTCTTMCCTAGGCCGATC3′ BstXTRA3D:5′TTGCAGAACCAATTTAATGGCGGCCAGTCAGGCCATCGTCG3′RT-PCR cloning was achievedwith kits from Clontech or Life Technologies. The gel-purified RT-PCRproducts were sequenced. The purified RT-PCR fragments were alsodigested for subcloning. Dominant negative ZAP70 (KI) and ZAP70SH2 (N+C)as well as selected hits from cDNA screens were subcloned to theretroviral pTRA-IRES-GFP vector. Selected hits form cDNA screens werealso subcloned to CRU5-IRES-GFP for infection of human primary Tlymphocytes.

Equivalents

[0367] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

We claim:
 1. A method of screening for cells having an alteredphenotype, the method comprising the steps of: a) providing a populationof cells having a parent phenotype, said population of cells comprisinga nucleic acid encoding a first element expressed in said population ofcells; b) introducing into said population of cells a library of fusionnucleic acids, said fusion nucleic acids each comprising: (1) a secondelement that is regulatable by said first element; and (2) a nucleicacid sequence encoding a candidate bioactive agent, wherein said nucleicacid sequence is operably linked to said second element; c) inducing theexpression of said nucleic acid sequence by contacting a third elementwith said population of cells; d) collecting a first subpopulation ofcells having an altered phenotype; e) repressing the expression of saidnucleic acid sequence by modulating said contacting of said thirdelement with said first subpopulation of cells; f) collecting a secondsubpopulation of cells having said parent phenotype; g) inducing theexpression of said nucleic acid sequence by contacting said thirdelement with said second subpopulation of cells; and 8) detecting athird subpopulation of cells having said altered phenotype.
 2. Themethod according to claim 1 further comprising: i) collecting said thirdsubpopulation of cells having said altered phenotype; j) repressing theexpression of said nucleic acid sequence by modulating said contactingof said third element with said third subpopulation of cells; and k)detecting a fourth subpopulation of cells having said parent phenotype.3. The method according to claim 2 further comprising: l) collectingsaid fourth subpopulation of cells having said parent phenotype; m)inducing the expression of said nucleic acid sequence by contacting saidthird element with said fourth subpopulation of cells; and n) detectinga fifth subpopulation of cells having said altered phenotype.
 4. Amethod of screening for cells having an altered phenotype, the methodcomprising the steps of: a) providing a population of cells having aparent phenotype, said population of cells comprising a nucleic acidencoding a first element; b) introducing into said population of cells alibrary of fusion nucleic acids, said fusion nucleic acids eachcomprising: (1) a second element that is regulatable by said firstelement; and (2) a nucleic acid sequence encoding a candidate bioactiveagent, wherein said nucleic acid sequence is operably linked to saidsecond element; c) inducing the expression of said nucleic acid sequenceby expressing said first element in said population of cells; d)collecting a first subpopulation of cells having an altered phenotype;e) repressing the expression of said nucleic acid by contacting a thirdelement with said first subpopulation of cells; f) collecting a secondsubpopulation of cells having said parent phenotype; g) inducing theexpression of said nucleic acid sequence by modulating said contactingof said third element with said second subpopulation of cells; and h)detecting a third subpopulation of cells having said altered phenotype.5. The method according to claim 4 further comprising: i) collectingsaid third subpopulation of cells having said altered phenotype; j)repressing the expression of said nucleic acid sequence by contactingsaid third element with said third subpopulation of cells; and k)detecting a fourth subpopulation of cells having said parent phenotype.6. The method according to claim 5 further comprising: l) collectingsaid fourth subpopulation of cells having said parent phenotype; m)inducing the expression of said nucleic acid sequence by modulating saidcontacting of said third element with said fourth subpopulation ofcells; and n) detecting a fifth subpopulation of cells having saidaltered phenotype.
 7. The method according to any of claims 1-3, whereinsaid first element comprises a reverse tetracycline-dependenttransactivator (rtTA).
 8. The method according to any of claims 4-6,wherein said first element comprises a tetracycline dependenttransactivator (tTA).
 9. The method according to any of claims 1-3 and4-6, wherein said second element comprises an tetracycline operatorsequence (TetO).
 10. The method according to any of claims 1-3 and 4-6,wherein said second element comprises an oligomer of a tetracyclineoperator sequence (TetO).
 11. The method according to any of claims 1-3and 4-6, wherein said third element comprises tetracycline (Tet). 12.The method according to any of claims 1-3 and 4-6, wherein said thirdelement comprises a tetracycline analogue.
 13. The method according toany of claims 1-3 and 4-6, wherein said third element comprisesdoxycycline (Tet).
 14. The method according to any of claims 1-3,wherein: said first element comprises a reverse tetracycline-dependentactivator (rtTA); said second element comprises an oligomer of atetracycline operator sequence (TetO); and said third element comprisestetracycline or doxycycline.
 15. The method according to any of claims4-6, wherein: said first element comprises a tetracycline-dependentactivator (rtTA); said second element comprises an oligomer of atetracycline operator sequence (TetO); and said third element comprisestetracycline (Tet) or doxycycline (Dox).
 16. The method according to anyof claims 1-3 and 4-6, wherein said population of cells comprise astimulator and said parent phenotype is due to the presence of saidstimulator.
 17. The method according to any of claims 1-3 and 4-6,wherein said fusion nucleic acids are each a component of a retroviralvector.
 18. The method according to any of claims 1-3 and 4-6, whereinsaid candidate bioactive agent is a polypeptide.
 19. The methodaccording to any of claims 1-3 and 4-6, wherein said candidate bioactiveagent is a cyclic polypeptide.
 20. The method according to any of claims1-3 and 4-6, wherein said first element is expressed stably ortransiently.
 21. The method according to any of claims 1-3 and 4-6,wherein said first is expressed constitutively.
 22. The method accordingto any of claims 1-3 and 4-6, wherein said first element is expressed intrans or in cis relative to said candidate bioactive agent.
 23. Themethod according to any of claims 1-3 and 4-6, wherein the expression ofsaid first element is inducible.
 24. The method according to any ofclaims 1-3 and 4-6, wherein said fusion nucleic acids comprise saidnucleic acid encoding said first element.
 25. The method according toany of claims 1-3 and 4-6, wherein said candidate bioactive agent is anRNA
 26. The method according to any of claims 1-3 and 4-6, wherein saidcandidate bioactive agent is an antisense RNA.
 27. The method accordingto any of claims 1-3 and 4-6, wherein said candidate bioactive agent isa DNA.
 28. The method according to any of claims 1-3 and 4-6, whereinsaid nucleic acid sequence comprises a full-length cDNA encoding saidcandidate bioactive agent.
 29. The method according to any of claims 1-3and 4-6, wherein said nucleic acid sequence comprises a subsequence of afull-length cDNA encoding said candidate bioactive agent.
 30. The methodaccording to any of claims 1-3 and 4-6, wherein said nucleic acidsequence comprises an antisense sequence of a full-length cDNA encodingsaid candidate bioactive agent.
 31. The method according to any ofclaims 1-3 and 4-6, wherein said nucleic acid sequence comprises anantisense sequence that is a subsequence of a full-length cDNA encodingsaid candidate bioactive agent.
 32. The method according to any ofclaims 1-3 and 4-6, wherein said nucleic acid sequence encodes an aminoacid sequence that is in-frame or out-of-frame as compared to the openreading frame (ORF) encoded by the amino acid sequence of a full-lengthcDNA, said amino acid sequence encoding said candidate bioactive agent.33. The method according to any of claims 1-3 and 4-6, wherein saidlibrary of fusion nucleic acids comprises about 10³ to 10⁹ differentsaid nucleic acid sequences.
 34. The method according to any of claims1-3 and 4-6, wherein said nucleic acid sequence is a random nucleic acidsequence.
 35. The method according to any of claims 1-3 and 4-6, whereinsaid nucleic acid sequence is a biased random nucleic acid sequence. 36.The method according to any of claims 1-3 and 4-6, wherein said librarycomprises about 10⁴ to 10⁸ different random nucleic acid sequences. 37.The method according to any of claims 1-3 and 4-6, wherein said fusionnucleic acid further comprises a sequence encoding a reporter protein,wherein said reporter protein is operably linked to said nucleic acidsequence.
 38. The method according to any of claims 1-3 and 4-6, whereinsaid fusion nucleic acid further comprises a sequence encoding areporter protein that is an auto fluorescent protein, wherein saidreporter protein is operably linked to said nucleic acid sequence. 39.The method according to any of claims 1-3 and 4-6, wherein said fusionnucleic acid further comprises a sequence encoding a reporter proteinthat is green fluorescent protein (GFP), wherein said reporter proteinis operably linked to said nucleic acid sequence.
 40. The methodaccording to any of claims 1-3 and 4-6, wherein said fusion nucleic acidfurther comprises a sequence encoding a reporter protein that is greenfluorescent protein (GFP) from Aqueorea, wherein said reporter proteinis operably linked to said nucleic acid sequence.
 41. The methodaccording to any of claims 1-3 and 4-6, wherein said fusion nucleic acidfurther comprises a sequence encoding a reporter protein that is greenfluorescent protein (GFP) from a Renilla species, wherein said reporterprotein is operably linked to said nucleic acid sequence.
 42. The methodaccording to any of claims 1-3 and 4-6, wherein said collecting is byfluorescence-activated cell sorting (FACS).
 43. The method according toany of claims 1-3 and 4-6, wherein said fusion nucleic acid furthercomprises a sequence encoding a reporter protein that is greenfluorescent protein (GFP) from a Renilla species, wherein said reporterprotein is operably linked to said nucleic acid sequence; and whereinsaid collecting is by fluorescence-activated cell sorting (FACS)
 44. Themethod according to any of claims 1-3 and 4-6, wherein said cells ofsaid population are mammalian cells.
 45. The method according to any ofclaims 1-3 and 4-6, wherein said altered phenotype comprises themodulation of cell cycle regulation due to the presence of saidcandidate bioactive agent.
 46. The method according to any of claims 1-3and 4-6, wherein said altered phenotype comprises the modulation ofexocytosis due to the presence of said candidate bioactive agent. 47.The method according to any of claims 1-3 and 4-6, wherein said alteredphenotype comprises the modulation of exocytosis due to the presence ofsaid candidate bioactive agent.
 48. The method according to any ofclaims 1-3 and 4-6, wherein said altered phenotype comprises themodulation of IgE synthesis due to the presence of said candidatebioactive agent.
 49. The method according to any of claims 1-3 and 4-6,wherein said altered phenotype comprises the modulation of IgE secretiondue to the presence of said candidate bioactive agent.
 50. The methodaccording to any of claims 1-3 and 4-6, wherein said altered phenotypecomprises the modulation of antigen-induced B cell differentiation dueto the presence of said candidate agent.
 51. The method according to anyof claims 1-3 and 4-6, wherein said altered phenotype comprises themodulation of antigen-induced B cell isotype switching due the presenceof said candidate agent.
 52. The method according to any of claims 1-3and 4-6, wherein said altered phenotype comprises the modulation of IgEswitching due to the presence of said candidate bioactive agent.
 53. Themethod according to any of claims 1-3 and 4-6, wherein said alteredphenotype comprises the modulation of apoptosis due to the presence ofsaid candidate bioactive agent.
 54. The method according to any ofclaims 1-3 and 4-6, wherein said altered phenotype comprises themodulation of angiogenesis due to the presence of said candidatebioactive agent.
 55. The method according to any of claims 1-3 and 4-6,wherein said altered phenotype comprises the modulation of T cellreceptor (TCR) activation due to the presence of said candidatebioactive agent.
 56. The method according to any of claims 1-3 and 4-6,wherein said altered phenotype comprises the modulation of a T cellsurface marker due to the presence of said candidate bioactive agent,wherein said marker is selected from a group of markers consisting ofCD3, CD25, CD28, CD40L, CD69, CD95, and CD95L.